Compounds, compositions and methods of treating cancer and fibrotic diseases

ABSTRACT

The present invention provides antibodies or antigen-binding fragments thereof that specifically hind the ENDO180 polypeptide and are internalized thereby, to conjugates comprising the molecules, to compositions comprising the antibodies and conjugates and to methods of using the same for delivery of therapeutic agents to cells that express the ENDO180 polypeptide on the surface of the cell for treating cell proliferative diseases or disorders and fibrosis, and for controlling (modulating) tumor progression.

RELATED APPLICATION

This application claims priority of U.S. Provisional Patent ApplicationNo. 61/162,348 filed Mar. 23, 2009 and which is hereby incorporated byreference herein in its entirety.

FIELD OF THE INVENTION

The present invention relates to molecules that target the ENDO180polypeptide and are internalized thereby, to conjugates comprising themolecules, to compositions comprising the molecules and conjugates andto methods of using the same for delivery of therapeutic agents to cellsthat express an ENDO180 polypeptide on the surface of the cell fortreating cell proliferative diseases or disorders and fibrosis, and forcontrolling (modulating) tumor progression.

BACKGROUND OF THE INVENTION ENDO180 Receptor

ENDO180, also known as CD280, uPARAP (urokinase plasminogen activatorreceptor associated protein) and mannose receptor C type 2 (MRC2), is arecycling endocytic receptor that directs bound ligands to degradationin the endosomes. It is part of a triple complex with urokinase typeplasmin activator (uPA) and urokinase-type plasmin activator receptor(uPAR), thus being involved in the production of plasmin fromplasminogen. Plasmin, in turn, is known to play a role in bothextracellular matrix (ECM) turnover and proteolytic conversion of latentTGF-beta into its active form.

In addition to its role in the production of plasmin, the triple complexwas shown to be involved in the activation of matrix metalloproteinase(MMP) proenzymes, to act on fibrin to bind several collagens and ingeneral turnover of extracellular matrix. This complex also takes partin cell adhesion and signal transduction (Bherendt et al, 2000. JBC275:1993-2002).

ENDO180 is a recycling endocytic receptor that functions in cellmotility and remodeling of the extracellular matrix by promoting cellmigration and uptake of collagens for intracellular degradation (Niels.2004 Biol Chem. 385(2):103-36; Kjoller et al, 2004 Exp Cell Res.293(1):106-16; Wienke et al., 2007 Cancer Res. 67(21):10230-40). ENDO180shares homology with the macrophage mannose receptor family: mannosereceptor, phosphlipase A₂ and DEC-205/MR6 (Isacke et al., 1990 Mol.Cell. Biol. 10:2606-2618; Sheikh et al., 2000, J. Cell. Sci. 113:1021-1032; Behrendt et al., 2000, J. Biol. Chem. 275: 1993-2002). Thisfamily grouping is based on an overall structural conservation: a largeextracellular domain comprising an N-terminal signal sequence followedby a cysteine-rich domain, a fibronectin type II domain (FNII), and 8 or10 C-type lectin-like domains (CTLDs) and small transmembrane andintracellular domains (˜66 amino acids together). As a family, thesereceptors have two striking features: First, although they belong to thelarge C-type lectin superfamily, they uniquely contain multiple CTLDswithin a single polypeptide backbone (Taylor M. E., 1997 Glycobiology 7:v-vii; McKay et al, 1998, Eur. J. Immunol. 28: 4071-4083; Howard andIsacke, 2002, supra). Second, they share the ability to be recycledbetween the plasma membrane and intercellular compartments of the cell(Isacke et al, 1990, supra; Zvaritch et al., 1996, J. Biol. Chem. 271:250-257). ENDO180 is unusual in the family of mannose receptors in thatit is targeted from the plasma membrane to the recycling endosomesrather than to a late endosome/lysosome compartment (Howard and Isacke,2002 supra).

ENDO180 is localized on the cell surface, in clathrin coated pits(Isacke et al., 1990 Mol. Cell. Biol. 10: 2606-2618; Sheikh et al.,2000, J. Cell. Sci. 113: 1021-1032) and in endosomes. It is mainlyexpressed in fibroblasts, endothelial cells and macrophages. In situhybridization showed its expression in highly vascularized organs.ENDO180 has also been found in bone-forming regions in mouse embryos (Wuet al., 1996, J. Biol. Chem. 271:21323-21330), and in osteoblasts andosteocytes at sites of endochondral and intramembraneous ossificationduring development (Engelholm et al., 2001, Trends Cardiovasc. Med.11:7-13.

The following patent publications also relate to the ENDO180 receptor:U.S. Pat. No. 6,117,977; U.S. Pat. No. 7,399,468; WO 97/40154 and WO00/58473. PCT Patent Publication No. WO 2004/100759 and US PatentPublication Nos. 2007/0072244 and 2009/0202566 to the assignee of thepresent invention and hereby incorporated by reference in their entiretyrelate to methods of identifying compounds capable of modulating humanENDO180 receptor activity.

Antibody Therapy

The search for new therapies to treat cancer and other diseases hasresulted in the development of human and humanized antibodies capable ofinhibiting receptor function. International patent publication WO2006/023491 provides a method of RNA interference, which comprisescontacting the cell with a fusion protein-double stranded RNA complex,the complex comprising the double stranded RNA segment containing adouble stranded RNA of interest and a fusion protein which is anantibody Fab fragment-protamine fusion protein.

SUMMARY OF THE INVENTION

The present invention is based in part on the identification of isolatedmolecules that specifically bind the ENDO180 polypeptide on a cellsurface. In some embodiments the molecules bind the extracellular domainof the ENDO180 polypeptide and are internalized into the cell by thepolypeptide, thereby providing a vehicle useful for delivery oftherapeutic and diagnostic cargo to a cell expressing the ENDO180polypeptide. Accordingly, in some embodiments the present inventionprovides a conjugate comprising a molecule that specifically binds theENDO180 polypeptide and a therapeutic agent useful for the delivery ofthe therapeutic agent into the cell. In some embodiments the ENDO180polypeptide is substantially identical to an amino acid sequence setforth in SEQ ID NO:2, encoded by a polynucleotide substantiallyidentical to a nucleic acid sequence set forth in SEQ ID NO:1.

In one aspect the present invention provides an anti-ENDO180 antibodywhich is produced by hybridoma cell line designated E3-8D8 (BCCMAccession Number LMBP 7203CB), or a fragment of the antibody, whichbinds to ENDO180 receptor on the surface of a cell. In some embodimentsbinding of the antibody to the receptor results in internalization ofthe antibody into the cell. Also provided is the E3-8D8 hybridoma cellline.

In some embodiments the antibody or fragment thereof is humanized or achimeric antibody or fragment thereof.

The invention provides a composition comprising at least oneanti-ENDO180 antibody or fragment thereof, the antibody produced by theE3-8D8 hybridoma or a humanized molecule thereof a chimeric antibody orfragment thereof, together with a carrier.

In some embodiments the isolated antibody is selected from the groupconsisting of a full IgG, a Fab fragment, a Fab′ fragment, an F(ab′)2fragment, the variable portion of the heavy and/or light chains thereof,Fab miniantibodies, and a scFv. In some embodiments the antibody is arecombinant polypeptide comprising a heavy chain CDR3 domain having anamino acid sequence set forth in SEQ ID NO:7 or a variant thereof whichretains the ability to specifically bind ENDO180. In some embodimentsthe antibody further comprises a light chain CDR3 domain having an aminoacid sequence set forth in SEQ ID NO:8 or a variant thereof whichretains the ability to specifically bind ENDO180.

In some embodiments the antibody is a scFv recombinant polypeptidecomprising an amino acid sequence set forth in SEQ ID NO:6 or a variantthereof, which retains the ability to specifically bind ENDO180. Inspecific embodiments the antibody exhibiting binding affinity to ENDO180receptor and comprising CDR3 domains set forth in SEQ ID NOS 7 and 8 isinternalized by the receptor into the cell expressing ENDO180 uponcontact of the antibody to the receptor.

The invention further provides a composition comprising at least oneanti-ENDO180 antibody or fragment thereof, as described above, and amoiety including a radioisotope, a therapeutic agent, a cytotoxic agent,or a detectable label. In some embodiments the moiety is attached (orlinked, or conjugated), either covalently, through a linker or achemical bond, or noncovalently, through ionic, van der Waals,electrostatic, or hydrogen bonds, to the antibody.

In some embodiments provided is an anti-ENDO180 antibody orantigen-binding fragment thereof selected from

-   -   a) the monoclonal antibody produced by the hybridoma cell line        E3-8D8 (BCCM Accession Number LMBP 7203CB);    -   b) an antibody or fragment thereof that binds to the same        epitope as the antibody in (a);    -   c) a humanized antibody of (a) or (b);    -   d) a fragment of an antibody comprising a polypeptide        substantially similar to SEQ ID NO: 6; and    -   e) a recombinant polypeptide comprising CDR3 with an amino acid        sequence substantially similar to amino acid sequences set forth        in SEQ ID NO:7 and 8.

Further provided is a composition comprising an anti-ENDO180 antibody orantigen-binding fragment thereof selected from

-   -   a) the monoclonal antibody produced by the hybridoma cell line        E3-8D8 (BCCM Accession Number LMBP 7203CB);    -   b) an antibody or fragment thereof that binds to the same        epitope as the antibody in (a);    -   c) a humanized antibody of (a) or (b);    -   d) a fragment of an antibody comprising a polypeptide        substantially similar to SEQ ID NO: 6; and    -   e) a recombinant polypeptide comprising CDRs having an amino        acid sequence substantially similar to amino acid sequences set        forth in SEQ ID NO:7 and 8.

In some embodiments the composition further comprises a moiety includinga radioisotope, a therapeutic agent, a cytotoxic agent, or a detectablelabel.

The present invention also provides a method of treating a subjectafflicted with a proliferative disorder comprising administering to thesubject a composition comprising an anti-ENDO180 antibody orantigen-binding fragment thereof selected from

-   -   a) the monoclonal antibody produced by the hybridoma cell line        E3-8D8 (BCCM Accession Number LMBP 7203CB);    -   b) an antibody or fragment thereof that binds to the same        epitope as the antibody in (a);    -   c) a humanized antibody of (a) or (b);    -   d) a fragment of an antibody comprising a polypeptide        substantially similar to SEQ ID NO: 6; and    -   e) a recombinant polypeptide comprising CDRs having an amino        acid sequence substantially similar to amino acid sequences set        forth in SEQ ID NO:7 and 8.

In some embodiments the proliferative disorder is selected from a solidtumor, a hematopoietic tumor, metastases, fibrosis and a macrophageassociated disorder.

In some embodiments the tumor is an ovarian tumor, a breast tumor,osteoblastic/osteocytic cancer, prostate cancer, head and neck cancer,leukemia, renal cell carcinoma, or transitional cell carcinoma.

In some embodiments the fibrosis is liver fibrosis, myelofibrosis,kidney fibrosis for any reason (CKD including end-stage renal disease,ESRD); lung fibrosis (including interstitial lung fibrosis ILF);abnormal scarring (keloids) associated with all possible types of skininjury accidental and jatrogenic (operations); scleroderma;cardiofibrosis, failure of glaucoma filtering operation; intestinaladhesions.

In some embodiments the macrophage-associated disorder is inflammationor atherosclerosis.

In one aspect the present invention provides a conjugate comprising:

-   -   a) an antibody or an antigen binding portion thereof which        specifically binds to the extracellular domain of the ENDO180        polypeptide on the surface of a cell;    -   b) a moiety including a radioisotope, a therapeutic agent, a        cytotoxic agent, or a detectable label; and    -   c) optionally a linking moiety which links (a) to (b).

In some embodiments the moiety is a therapeutic agent selected from anoligonucleotide agent and a non-oligonucleotide agent. In someembodiments the therapeutic agent is an oligonucleotide therapeuticagent, including an inhibitory oligonucleotide. Accordingly, in variousembodiments the therapeutic agent is selected from an antisensecompound, a chemically modified siRNA compound, an unmodified siRNAcompound, a chemically modified shRNA compound, an unmodified shRNAcompound, a chemically modified miRNA compound, and an unmodified miRNAcompound. In various preferred embodiments the therapeutic agent ischemically modified siRNA. In some embodiments the chemically modifiedsiRNA compound inhibits expression of a target gene associated withcancer, fibrosis or macrophage associated disease. In some embodimentsthe target gene is selected from any one of the target genes set forthin Table A, hereinbelow.

In certain embodiments the therapeutic agent is attached to the antibodyvia a nucleotide or non-nucleotide linking moiety.

In yet another aspect the present invention provides a pharmaceuticalcomposition comprising the conjugate of the present invention.

In yet another aspect the present invention provides a method oftreating a subject suffering from a proliferative disease comprisingadministering to the subject a therapeutically effective amount of anantibody that specifically binds ENDO180 polypeptide and is internalizedby the ENDO180 polypeptide, wherein the antibody is covalently ornon-covalently bound to a therapeutic agent.

In some embodiments the proliferative disease is selected from malignantand benign proliferative disease. In some embodiments proliferativedisease is cancer. In other embodiments proliferative disease isfibrosis. Non-limiting examples of diseases and disorders for use of thepresent invention include

1. soft tissue sarcomas in which ENDO180 is expressed in the tumor andtumor stroma cells (activated myofibroblasts, neovasculature andinfiltrating cells of macrophage-monocyte lineage);2. carcinomas in which ENDO180 is expressed in the tumor stroma cells(activated myofibroblasts, neovasculature and infiltrating cells ofmacrophage-monocyte lineage);3. carcinoma that express ENDO180 and have undergoneepithelial-mesenchymal transition thus acquiring high metastaticpotential;4. leukemia expressing ENDO180 for example, from macrophage-monocytelineage;5. fibrotic diseases, for example of kidney, lung and liver withactivated myofibroblasts;6. diseases and disorders associated with macrophage includingatherosclerosis and chronic inflammation.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A-1J provides polynucleotide and amino acid sequences of variouscompounds according to the present invention. FIG. 1A: human ENDO180mRNA (SEQ ID NO:1); FIG. 1B: human ENDO180 polypeptide (SEQ ID NO:2);FIG. 1C: SEQ ID NO:3 polynucleotide sequence of extracellular domain ofhuman ENDO180 (amino acids 1-522) with FLAG sequence, FLAG domainunderlined (pcDNA3-5′ hendo180-FLAG construct, SEQ ID NO:3); FIG. 1Dpolypeptide sequence of SEQ ID NO:3 (SEQ ID NO:4); FIG. 1E:polynucleotide sequence of scFv clone G7V (SEQ ID NO:5); FIG. 1F:polypeptide sequence of scFv clone G7V (SEQ ID NO:6); FIG. 1G. heavychain CDR3 of G7V (SEQ ID NO:7); FIG. 1H. light chain CDR3 of G7V (SEQID NO:8); FIG. 1J: polypeptide 1-522 of the extracellular domain ofhuman ENDO180.

FIGS. 2A-2H. Internalization of CypHer5E fluorophore anti-ENDO180 mAbsto ENDO180 expressing cells.

FIG. 3. Internalization of Biotin anti-ENDO180 mAbs to mice havingUnilateral Ureter Obstructed kidney.

FIG. 4. Internalization of anti-ENDO180 mAbs conjugated to CypHer5Efluorophore into Myelo-Monocytoid human leukemia MonoMac cell lineexpressing ENDO180.

DETAILED DESCRIPTION OF THE INVENTION Definitions

For convenience certain terms employed in the specification, examplesand claims are described herein.

It is to be noted that, as used herein, the singular forms “a”, “an” and“the” include plural forms unless the content clearly dictatesotherwise.

Where aspects or embodiments of the invention are described in terms ofMarkush groups or other grouping of alternatives, those skilled in theart will recognize that the invention is also thereby described in termsof any individual member or subgroup of members of the group.

An “inhibitor” is a compound, which is capable of reducing (partially orfully) the expression of a gene or the activity of the product of suchgene to an extent sufficient to achieve a desired biological orphysiological effect. The term “inhibitor” as used herein includes oneor more of an oligonucleotide inhibitor, including siRNA, shRNA,synthetic shRNA; miRNA, antisense RNA and DNA and ribozymes. An“inhibitory oligonucleotide” includes an antisense compound, achemically modified siRNA compound, an unmodified siRNA compound, achemically modified shRNA compound, an unmodified shRNA compound, achemically modified miRNA compound, and an unmodified miRNA compound.

A “siRNA inhibitor” is a compound which is capable of reducing theexpression of a gene or the activity of the product of such gene to anextent sufficient to achieve a desired biological or physiologicaleffect. The term “siRNA inhibitor” as used herein refers to one or moreof a siRNA, shRNA, synthetic shRNA; miRNA. Inhibition may also bereferred to as down-regulation or, for RNAi, silencing.

The term “inhibit” as used herein refers to reducing the expression of agene or the activity of the product of such gene to an extent sufficientto achieve a desired biological or physiological effect. Inhibition maybe complete or partial. As used herein, the term “ENDO180 gene” isdefined as any homolog of the ENDO180 gene having preferably 90%homology, more preferably 95% homology, and even more preferably 98%homology to the amino acid encoding region of SEQ ID NO:1 or nucleicacid sequences which bind to the ENDO180 gene under conditions of highlystringent hybridization, which are well-known in the art (for example,see Ausubel et al., Current Protocols in Molecular Biology, John Wileyand Sons, Baltimore, Md. (1988), updated in 1995 and 1998).

As used herein, the term “ENDO180” or “ENDO180 polypeptide” or “ENDO180receptor” is defined as any homolog of the ENDO180 polypeptide havingpreferably at least 90% homology, more preferably at least 95% homology,and even more preferably at least 98% homology or 100% identity to SEQID NO:2, as either full-length or a fragments or a domain thereof, as amutant or the polypeptide encoded by a spliced variant nucleic acidsequence, as a chimera with other polypeptides, provided that any of theabove has the same or substantially the same biological function as theENDO180 receptor. ENDO180 polypeptide, or an ENDO180 polypeptidehomolog, may be present in different forms, including but not limited tosoluble protein, membrane-bound (either in purified membranepreparations or on a cell surface), bead-bound, or any other formpresenting ENDO180 protein or fragments and polypeptides derivedthereof. The term “inhibit” as used herein refers to reducing theexpression of a gene or the activity of the product of such gene to anextent sufficient to achieve a desired biological or physiologicaleffect. Inhibition is either complete or partial.

The terms “mRNA polynucleotide sequence”, “mRNA sequence” and “mRNA” areused interchangeably.

As used herein, the terms “polynucleotide” and “nucleic acid” may beused interchangeably and refer to nucleotide sequences comprisingdeoxyribonucleic acid (DNA), and ribonucleic acid (RNA). The terms areto be understood to include, as equivalents, analogs of either RNA orDNA made from nucleotide analogs. Throughout this application, mRNAsequences are set forth as representing the corresponding genes.

“Oligonucleotide” or “oligomer” refers to a deoxyribonucleotide orribonucleotide sequence from about 2 to about 50 nucleotides. Each DNAor RNA nucleotide may be independently natural or synthetic, and ormodified or unmodified. Modifications include changes to the sugarmoiety, the base moiety and or the linkages between nucleotides in theoligonucleotide. The compounds of the present invention encompassmolecules comprising deoxyribonucleotides, ribonucleotides, modifieddeoxyribonucleotides, modified ribonucleotides and combinations thereof.

Substantially complementary refers to complementarity of greater thanabout 84%, to another sequence. For example in a duplex regionconsisting of 19 base pairs one mismatch results in 94.7%complementarity, two mismatches results in about 89.5% complementarityand 3 mismatches results in about 84.2% complementarity, rendering theduplex region substantially complementary. Accordingly substantiallyidentical refers to identity of greater than about 84%, to anothersequence.

The conjugate of the present invention comprises a) an antibody orfragment thereof, which specifically binds to an ENDO180 polypeptide onthe surface of a cell, b) a nucleotide-based therapeutic agent selectedfrom an antisense compound, a chemically modified siRNA compound, anunmodified siRNA compound, a chemically modified shRNA compound, anunmodified shRNA compound, a chemically modified miRNA compound, and anunmodified miRNA compound; and c) a linking moiety which links (a) to(b); wherein the nucleotide-based therapeutic agent inhibits expressionof the target gene in the cell.

The “linker” according to the present invention is a nucleotide ornon-nucleotide moiety which links the antibody to the therapeuticmolecule. In some embodiments the linker is a cleavable moiety.Preferred cleavable groups include a disulfide bond, amide bond,thioamide, bond, ester bond, thioester bond, vicinal diol bond, orhemiacetal. Other cleavable bonds include enzymatically-cleavable bonds,such as peptide bonds (cleaved by peptidases), phosphate bonds (cleavedby phosphatases), nucleic acid bonds (cleaved by endonucleases), andsugar bonds (cleaved by glycosidases).

In some embodiments the linker is a non-nucleotide linker including apeptide linker. The choice of peptide sequence is critical to thesuccess of the conjugate. In some embodiments the linker is stable toserum proteases, yet is cleaved by the lysosomal enzymes in the targetcell. In a non-limiting example the linker is a peptide selected from alinker set forth in U.S. Pat. No. 5,574,142, protamine, a fragment ofprotamine, (Arg)9, biotin-avidin, biotin-streptavidin and antennapediapeptide. For example, a peptide linker is used to link the antibody to anucleotide therapeutic agent. Other non-nucleotide linkers include alkylor aryl chains of about 5 to about 100 atoms.

In some embodiments the linker is a nucleotide linker. In certainembodiments a nucleic acid linker has a length ranging from 2-100,preferably 2-50 or 2-30 nucleotides.

Oligonucleotide Chemical Modifications

“Nucleotide” is meant to encompass deoxyribonucleotides andribonucleotides, which may be natural or synthetic, and or modified orunmodified. Modifications include changes to the sugar moiety, the basemoiety and or the linkages between ribonucleotides in theoligoribonucleotide. As used herein, the term “ribonucleotide”encompasses natural and synthetic, unmodified and modifiedribonucleotides. Modifications include changes to the sugar moiety, tothe base moiety and/or to the linkages between ribonucleotides in theoligonucleotide.

The nucleotides useful in preparing a therapeutic agent includenaturally occurring or synthetic modified bases. Naturally occurringbases include adenine, guanine, cytosine, thymine and uracil. Modifiedbases of nucleotides include inosine, xanthine, hypoxanthine,2-aminoadenine, 6-methyl, 2-propyl and other alkyl adenines, 5-halouracil, 5-halo cytosine, 6-aza cytosine and 6-aza thymine, pseudouracil, 4-thiouracil, 8-halo adenine, 8-aminoadenine, 8-thiol adenine,8-thiolalkyl adenines, 8-hydroxyl adenine and other 8-substitutedadenines, 8-halo guanines, 8-amino guanine, 8-thiol guanine, 8-thioalkylguanines, 8-hydroxyl guanine and other substituted guanines, other azaand deaza adenines, other aza and deaza guanines, 5-trifluoromethyluracil and 5-trifluoro cytosine. In some embodiments one or morenucleotides in an oligomer is substituted with inosine.

According to some embodiments the present invention provides inhibitoryoligonucleotide compounds comprising unmodified and modified nucleotidesand or unconventional moieties. In certain embodiments the therapeuticagent is an oligonucleotide. In various preferred embodiments thetherapeutic agent is a double stranded oligonucleotide and preferablysiRNA.

The selection and synthesis of siRNA corresponding to known genes hasbeen widely reported; (see for example Ui-Tei et al., 2006. J BiomedBiotechnol.; 2006:65052; Chalk et al., 2004. BBRC. 319(1): 264-74; Sioud& Leirdal, 2004. Met. Mol. Biol.; 252:457-69; Levenkova et al., 2004,Bioinform. 20(3):430-2; Ui-Tei et al., 2004. NAR 32(3):936-48).

For examples of the use of, and production of, modified siRNA see forexample Braasch et al., 2003. Biochem., 42(26):7967-75; Chiu et al.,2003, RNA, 9(9):1034-48; PCT publications WO 2004/015107 (atugen AG) andWO 02/44321 (Tuschl et al). U.S. Pat. Nos. 5,898,031 and 6,107,094 teachchemically modified oligomers. U.S. Pat. No. 7,452,987 relates tooligomeric compounds having alternating unmodified and 2′ sugar modifiedribonucleotides. US patent publication No. 2005/0042647 describes dsRNAcompounds having chemically modified internucleotide linkages.

Amarzguoui et al., (2003, NAR, 31(2):589-595) showed that siRNA activitydepended on the positioning of the 2′-O-methyl modifications. Holen etal (2003, NAR, 31(9):2401-2407) report that an siRNA having smallnumbers of 2′-O-methyl modified nucleosides showed good activitycompared to wild type but that the activity decreased as the numbers of2′-O-methyl modified nucleosides was increased. Chiu and Rana (2003,RNA, 9:1034-1048) teach that incorporation of 2′-O-methyl modifiednucleosides in the sense or antisense strand (fully modified strands)severely reduced siRNA activity relative to unmodified siRNA. Theplacement of a 2′-O-methyl group at the 5′-terminus on the antisensestrand was reported to severely limit activity whereas placement at the3′-terminus of the antisense and at both termini of the sense strand wastolerated (Czauderna et al., 2003, NAR, 31(11), 2705-2716).

PCT Patent Application Nos. PCT/IL2008/000248 and PCT/IL2008/001197,assigned to the assignee of the present invention and herebyincorporated by reference in their entirety disclose motifs useful inthe preparation of chemically modified siRNA compounds. PCT PatentPublication No. WO 2008/020435 discloses inhibitors, including somesiRNA compounds to the target genes set forth herein.

The compound comprises at least one modified nucleotide selected fromthe group consisting of a sugar modification, a base modification and aninternucleotide linkage modification and may contain DNA, and modifiednucleotides such as LNA (locked nucleic acid), ENA (ethylene-bridgednucleic acid), PNA (peptide nucleic acid), arabinoside,phosphonocarboxylate or phosphinocarboxylate nucleotide (PACEnucleotide), mirror nucleotide, or nucleotides with a 6 carbon sugar.

All analogs of, or modifications to, a nucleotide/oligonucleotide areemployed with the present invention, provided that said analog ormodification does not substantially adversely affect the function of thenucleotide/oligonucleotide. Acceptable modifications includemodifications of the sugar moiety, modifications of the base moiety,modifications in the internucleotide linkages and combinations thereof.

A sugar modification includes a modification on the 2′ moiety of thesugar residue and encompasses amino, fluoro, alkoxy e.g. methoxy, alkyl,amino, fluoro, chloro, bromo, CN, CF, imidazole, carboxylate, thioate,C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkaryl or aralkyl,OCF₃, OCN, O—, S—, or N-alkyl; O-, S, or N-alkenyl; SOCH₃; SO₂CH₃; ONO₂;NO₂, N₃; heterozycloalkyl; heterozycloalkaryl; aminoalkylamino;polyalkylamino or substituted silyl, as, among others, described inEuropean patents EP 0 586 520 B1 or EP 0 618 925 B1.

In one embodiment the siRNA compound comprises at least oneribonucleotide comprising a 2′ modification on the sugar moiety (“2′sugar modification”). In certain embodiments the compound comprises2′O-alkyl or 2′-fluoro or 2′O-allyl or any other 2′ modification,optionally on alternate positions. Other stabilizing modifications arealso possible (e.g. terminal modifications). In some embodiments apreferred 2′O-alkyl is 2′O-methyl (methoxy) sugar modification.

In some embodiments the backbone of the oligonucleotides is modified andcomprises phosphate-D-ribose entities but may also containthiophosphate-D-ribose entities, triester, thioate, 2′-5′ bridgedbackbone (also may be referred to as 5′-2′), PACE and the like.

As used herein, the terms “non-pairing nucleotide analog” means anucleotide analog which comprises a non-base pairing moiety includingbut not limited to: 6 des amino adenosine (Nebularine), 4-Me-indole,3-nitropyrrole, 5-nitroindole, Ds, Pa, N3-Me ribo U, N3-Me riboT, N3-MedC, N3-Me-dT, N1-Me-dG, N1-Me-dA, N3-ethyl-dC, N3-Me dC. In someembodiments the non-base pairing nucleotide analog is a ribonucleotide.In other embodiments it is a deoxyribonucleotide. In addition, analogsof polynucleotides may be prepared wherein the structure of one or morenucleotide is fundamentally altered and better suited as therapeutic orexperimental reagents. An example of a nucleotide analog is a peptidenucleic acid (PNA) wherein the deoxyribose (or ribose) phosphatebackbone in DNA (or RNA is replaced with a polyamide backbone which issimilar to that found in peptides. PNA analogs have been shown to beresistant to enzymatic degradation and to have extended stability invivo and in vitro. Other modifications that can be made tooligonucleotides include polymer backbones, cyclic backbones, acyclicbackbones, thiophosphate-D-ribose backbones, triester backbones, thioatebackbones, 2′-5′ bridged backbone, artificial nucleic acids, morpholinonucleic acids, glycol nucleic acid (GNA), threose nucleic acid (TNA),arabinoside, and mirror nucleoside (for example,beta-L-deoxyribonucleoside instead of beta-D-deoxyribonucleoside).Examples of siRNA compounds comprising LNA nucleotides are disclosed inElmen et al., (NAR 2005, 33(1):439-447).

The compounds of the present invention can be synthesized using one ormore inverted nucleotides, for example inverted thymidine or invertedadenine (see, for example, Takei, et al., 2002, JBC 277(26):23800-06).

Other modifications include terminal modifications on the 5′ and/or 3′part of the oligonucleotides and are also known as capping moieties.Such terminal modifications are selected from a nucleotide, a modifiednucleotide, a lipid, a peptide, a sugar and inverted abasic moiety.

What is sometimes referred to in the present invention as an “abasicnucleotide” or “abasic nucleotide analog” is more properly referred toas a pseudo-nucleotide or an unconventional moiety. A nucleotide is amonomeric unit of nucleic acid, consisting of a ribose or deoxyribosesugar, a phosphate, and a base (adenine, guanine, thymine, or cytosinein DNA; adenine, guanine, uracil, or cytosine in RNA). A modifiednucleotide comprises a modification in one or more of the sugar,phosphate and or base. The abasic pseudo-nucleotide lacks a base, andthus is not strictly a nucleotide.

In some embodiments the siRNA therapeutic agent comprises a cappingmoiety. The term “capping moiety” as used herein includes abasic ribosemoiety, abasic deoxyribose moiety, modifications abasic ribose andabasic deoxyribose moieties including 2′ O alkyl modifications; invertedabasic ribose and abasic deoxyribose moieties and modifications thereof;C6-imino-Pi; a mirror nucleotide including L-DNA and L-RNA; 5′O-Menucleotide; and nucleotide analogs including 4′,5′-methylene nucleotide;1-(β-D-erythrofuranosyl)nucleotide; 4′-thio nucleotide, carbocyclicnucleotide; 5′-amino-alkyl phosphate; 1,3-diamino-2-propyl phosphate,3-aminopropyl phosphate; 6-aminohexyl phosphate; 12-aminododecylphosphate; hydroxypropyl phosphate; 1,5-anhydrohexitol nucleotide;alpha-nucleotide; threo-pentofuranosyl nucleotide; acyclic 3′,4′-seconucleotide; 3,4-dihydroxybutyl nucleotide; 3,5-dihydroxypentylnucleotide, 5′-5′-inverted abasic moiety; 1,4-butanediol phosphate;5′-amino; and bridging or non bridging methylphosphonate and 5′-mercaptomoieties.

Certain preferred capping moieties are abasic ribose or abasicdeoxyribose moieties; inverted abasic ribose or abasic deoxyribosemoieties; C6-amino-Pi; a mirror nucleotide including L-DNA and L-RNA.

In some embodiments the therapeutic siRNA comprises a moiety other thana nucleotide. The term “unconventional moiety” as used herein refers toabasic ribose moiety, an abasic deoxyribose moiety, adeoxyribonucleotide, a modified deoxyribonucleotide, a mirrornucleotide, a non-base pairing nucleotide analog and a nucleotide joinedto an adjacent nucleotide by a 2′-5′ internucleotide phosphate bond;bridged nucleic acids including LNA and ethylene bridged nucleic acids.

Abasic deoxyribose moiety includes for example abasicdeoxyribose-3′-phosphate; 1,2-dideoxy-D-ribofuranose-3-phosphate;1,4-anhydro-2-deoxy-D-ribitol-3-phosphate. Inverted abasic deoxyribosemoiety includes inverted deoxyriboabasic; 3′,5′ inverted deoxyabasic5′-phosphate.

A “mirror” nucleotide is a nucleotide with reversed chirality to thenaturally occurring or commonly employed nucleotide, i.e., a mirrorimage (L-nucleotide) of the naturally occurring (D-nucleotide), alsoreferred to as L-RNA in the case of a mirror ribonucleotide, and“spiegelmer”. The nucleotide can be a ribonucleotide or adeoxyribonucleotide and my further comprise at least one sugar, base andor backbone modification. See U.S. Pat. No. 6,586,238. Also, U.S. Pat.No. 6,602,858 discloses nucleic acid catalysts comprising at least oneL-nucleotide substitution. Mirror nucleotide includes for example L-DNA(L-deoxyriboadenosine-3′-phosphate (mirror dA);L-deoxyribocytidine-3′-phosphate (mirror dC);L-deoxyriboguanosine-3′-phosphate (mirror dG);L-deoxyribothymidine-3′-phosphate (mirror image dT)) and L-RNA(L-riboadenosine-3′-phosphate (mirror rA); L-ribocytidine-3′-phosphate(mirror rC); L-riboguanosine-3′-phosphate (mirror rG);L-ribouracil-3′-phosphate (mirror dU).

Modified deoxyribonucleotide includes, for example 5′ OMe DNA(5-methyl-deoxyriboguanosine-3′-phosphate) which may be useful as anucleotide in the 5′ terminal position (position number 1); PACE(deoxyriboadenine 3′ phosphonoacetate, deoxyribocytidine 3′phosphonoacetate, deoxyriboguanosine 3′ phosphonoacetate,deoxyribothymidine 3′ phosphonoacetate.

Bridged nucleic acids include LNA (2′-0,4′-C-methylene bridged NucleicAcid adenosine 3′ monophosphate, 2′-0,4′-C-methylene bridged NucleicAcid 5-methyl-cytidine 3′ monophosphate, 2′-0,4′-C-methylene bridgedNucleic Acid guanosine 3′ monophosphate, 5-methyl-uridine (or thymidine)3′ monophosphate); and ENA (2′-0,4′-C-ethylene bridged Nucleic Acidadenosine 3′ monophosphate, 2′-0,4′-C-ethylene bridged Nucleic Acid5-methyl-cytidine 3′ monophosphate, 2′-0,4′-C-ethylene bridged NucleicAcid guanosine 3′ monophosphate, 5-methyl-uridine (or thymidine) 3′monophosphate).

In some embodiments of the present invention a preferred unconventionalmoiety is an abasic ribose moiety, an abasic deoxyribose moiety, adeoxyribonucleotide, a mirror nucleotide, and a nucleotide joined to anadjacent nucleotide by a 2′-5′ internucleotide phosphate bond.

According to one aspect the present invention provides inhibitoryoligonucleotide compounds comprising unmodified and modifiednucleotides. The compound comprises at least one modified nucleotideselected from the group consisting of a sugar modification, a basemodification and an internucleotide linkage modification and may containDNA, and modified nucleotides such as LNA (locked nucleic acid)including ENA (ethylene-bridged nucleic acid; PNA (peptide nucleicacid); arabinoside; PACE (phosphonoacetate and derivatives thereof),mirror nucleotide, or nucleotides with a six-carbon sugar. In someembodiments the present invention provides methods and compositions forinhibiting expression of a target gene in vivo. In general, the methodincludes administering a delivery-therapeutic agent conjugate. Inparticular embodiments small interfering RNAs (i.e. siRNAs), that targetan mRNA transcribed from the target gene in an amount sufficient todown-regulate expression (reduce mRNA, reduce protein levels) of atarget gene by an RNA interference mechanism. In particular, the subjectmethod can be used to inhibit expression of the target gene fortreatment of a disease. In accordance with the present invention, thesiRNA molecules or inhibitors of the target gene are used as drugs totreat various pathologies.

The synthesis of the nucleic acids described herein, is within theskills of the one of the art. Such synthesis is, among others, describedin Beaucage S L and Iyer R P, 1992 Tetrahedron; 48: 2223-2311, BeaucageS, and Iyer R P, 1993 Tetrahedron; 49: 6123-6194 and Caruthers M H et.al., 1987 Methods Enzymol.; 154: 287-313, the synthesis of thioates is,among others, described in Eckstein F., 1985 Annu Rev. Biochem.; 54:367-402, the synthesis of RNA molecules is described in Sproat B., inHumana Press 2005 Edited by Herdewijn P.; Kap. 2: 17-31 and respectivedownstream processes are, among others, described in Pingoud A. et. al.,in IRL Press 1989 Edited by Oliver R. W. A.; Kap. 7: 183-208 and SproatB., in Humana Press 2005 Edited by Herdewijn P.; Kap. 2: 17-31 (supra).siRNA for any one of the target genes is synthesized using methods knownin the art as described above, based on the known sequence of the targetgene mRNA and is stabilized to serum and/or cellular nucleases byvarious modifications as described herein.

Target Genes

The conjugates according to the present invention are useful forinhibiting expression of a gene associated with a disease or disorderselected from a proliferative disease a metastatic disease and fibrosis.

Target genes include anti-apoptotic genes, genes associated with basiccell division machinery, genes associated with cell cycleregulation/cell proliferation, genes associated with rate-limitingmetabolism (nucleotide/nucleic acid synthesis, protein synthesis, energymetabolism), genes associated with protein trafficking (e.g.,secretion); proinflammatory genes, cytokines, chemokines, NFkB, growthfactors/receptors (TGFβ1 and 2, CTGF, IGF1, PDGF1, PDGF2, VEGF, EGFR,HER2, etc).

A non-limiting list of target genes is set forth in Table A,hereinbelow.

Abbreviation full name AARSD1 alanyl-tRNA synthetase domain containing 1ABCF1 ATP-binding cassette, sub-family F (GCN20), member 1 AKT1 v-aktmurine thymoma viral oncogene homolog 1 AKT2 -akt murine thymoma viraloncogene homolog 2 AKT3 v-akt murine thymoma viral oncogene homolog 3(protein kinase B, ANG angiogenin, ribonuclease, RNase A family, 5 BADBCL2-associated agonist of cell death BAG1 BCL2-associated athanogeneBAK1 BCL2-antagonist/killer 1 BAX BCL2-associated X protein BCL2 B-cellCLL/lymphoma 2 BCL2A1 BCL2-related protein A1 BCL2L1 BCL2-like 1 BCL2L11BCL2-like 11 (apoptosis facilitator) BID BH3 interacting domain deathagonist CALR calreticulin CASP3 caspase 3, apoptosis-related cysteinepeptidase CASP9 caspase 9, apoptosis-related cysteine peptidase CASP9caspase 9, apoptosis-related cysteine peptidase CCNB1 cyclin B1 CD40CD40 molecule, TNF receptor superfamily member 5 CDC2 cell divisioncycle 2, G1 to S and G2 to M CDC73 cell division cycle 73, Paf1/RNApolymerase II complex component, CDH1 cadherin 1, type 1, E-cadherin(epithelial) CEBPB CCAAT/enhancer binding protein (C/EBP), beta CFLARCASP8 and FADD-like apoptosis regulator CHEK1 CHK1 checkpoint homolog(S. pombe) CMPK1 cytidine monophosphate (UMP-CMP) kinase 1, cytosolicCOL4A1 collagen, type IV, alpha 1 CTGF connective tissue growth factorDDIT4 DNA-damage-inducible transcript 4 DDIT4L DNA-damage-inducibletranscript 4 like EEF2K eukaryotic elongation factor-2 kinase EGFepidermal growth factor EIF2AK4 eukaryotic translation initiation factor2 alpha kinase 4 EPRS glutamyl-prolyl-tRNA synthetase ERBB2 -erb-b2erythroblastic leukemia viral oncogene homolog 2, ERBB3 v-erb-b2erythroblastic leukemia viral oncogene homolog 3 ESR1 estrogen receptor1 F3 coagulation factor III FAS Fas (TNF receptor superfamily, member 6)FEN1 flap structure-specific endonuclease 1 GAPDHglyceraldehyde-3-phosphate dehydrogenase H19 H19, imprinted maternallyexpressed transcript HDAC1 histone deacetylase 1 HGF hepatocyte growthfactor (hepapoietin A; scatter factor) HIF1A hypoxia inducible factor 1,alpha subunit HSF1 heat shock transcription factor 1 IER3 immediateearly response 3 IGF1 insulin-like growth factor 1 (somatomedin C) IGF1Rinsulin-like growth factor 1 receptor IGFBP5 insulin-like growth factorbinding protein 5 IL15 interleukin 15 IL8 interleukin 8 JUN iun oncogeneMADD MAP-kinase activating death domain MAPK1 mitogen-activated proteinkinase 1 MCL1 myeloid cell leukemia MDM2 Mdm2 p53 binding proteinhomolog (mouse) MIF macrophage migration inhibitory factor(glycosylation-inhibiting) MMP3 matrix metallopeptidase 3 (stromelysin1, progelatinase) MYC v-myc myelocytomatosis viral oncogene homolog(avian) c-MYC myelocytomatosis viral oncogene homolog (avian) NFKB1nuclear factor of kappa light polypeptide gene enhancer in B-cells 1NOS2 nitric oxide synthase 2, inducible NOTCH1 Notch homolog 1,translocation-associated (Drosophila) NOX1 NADPH oxidase 1 NOX2cytochrome b-245, beta polypeptide (CYBB) NOX3 NADPH oxidase 3 NOX4NADPH oxidase 4 NOX5 NADPH oxidase 5 NOXA1 NADPH oxidase activator 1NOXO1 NADPH oxidase organizer 1 NRF2 nuclear factor (erythroid-derived2)-like 2 NR4A1 nuclear receptor subfamily 4, group A, member 1 OAS12′,5′-oligoadenylate synthetase 1, 40/46 kDa OAS2 2′-5′-oligoadenylatesynthetase 2, 69/71 kDa OAS3 2′-5′-oligoadenylate synthetase 3, 100 kDaODC1 ornithine decarboxylase 1 PARP1 poly (ADP-ribose) polymerase 1 PCNAproliferating cell nuclear antigen PDGFA platelet-derived growth factoralpha polypeptide PIK3R1 phosphoinositide-3-kinase, regulatory subunit 1PLAU plasminogen activator, urokinase PLK1 polo-like kinase 1 POLA1polymerase (DNA directed), alpha 1, catalytic subunit POLD1 polymerase(DNA directed), delta 1, catalytic subunit 125 kDa POLE polymerase (DNAdirected), epsilon PPARD peroxisome proliferator-activated receptordelta PRKAR1A protein kinase, cAMP-dependent, regulatory, type I, alpha(tissue PRKDC protein kinase, DNA-activated, catalytic polypeptide PROK2prokineticin 2 PTK2 PTK2 protein tyrosine kinase 2 PTK2B PTK2B proteintyrosine kinase 2 beta RAC1 ras-related C3 botulinum toxin substrate 1(rho family, small GTP RASSF1 Ras association (RalGDS/AF-6) domainfamily member 1 REG1A regenerating islet-derived 1 alpha RFC3replication factor C (activator 1) 3, 38 kDa RHOA ras homolog genefamily, member A RPA1 replication protein A1, 70 kDa SIPA1signal-induced proliferation-associated 1 SOD1 superoxide dismutase 1,soluble SRC v-src sarcoma (Schmidt-Ruppin A-2) viral oncogene homolog(avian) STAT3 signal transducer and activator of transcription 3 STAT6signal transducer and activator of transcription 6, interleukin-4 TCF7L2transcription factor 7-like 2 TEK TEK tyrosine kinase, endothelialTFAP2B transcription factor AP-2 beta TGFβ1 transforming growth factor,beta 1 TIAF1 TGFB1-induced anti-apoptotic factor 1 TIMP1 TIMPmetallopeptidase inhibitor 1 TNF tumor necrosis factor TNFRSF1B tumornecrosis factor receptor superfamily, member 1B TP53 tumor protein p53TRAF1 TNF receptor-associated factor 1 TYMS thymidylate synthetase VEGFAvascular endothelial growth factor A XIAP X-linked inhibitor ofapoptosis

Sense and antisense sequences useful in the synthesis of siRNA areselected according to proprietary and publicly available methods andalgorithms.

The chemical modifications provided above are useful in synthesizingnucleotide therapeutics that exhibit inter alia, serum stability,activity, reduced immune response, reduced off target effect.

Antibodies

The term “antibody” refers to IgG, IgM, IgD, IgA, and IgE antibody,inter alia. The definition includes polyclonal antibodies or monoclonalantibodies. This term refers to whole antibodies or fragments ofantibodies comprising an antigen-binding domain, e.g. antibodies withoutthe Fc portion, single chain antibodies, miniantibodies, fragmentsconsisting of essentially only the variable, antigen-binding domain ofthe antibody, etc. The term “antibody” may also refer to antibodiesagainst polynucleotide sequences obtained by cDNA vaccination. The termalso encompasses antibody fragments which retain the ability toselectively bind with their antigen or receptor and are exemplified asfollows, inter alia:

(1) Fab, the fragment which contains a monovalent antigen-bindingfragment of an antibody molecule which can be produced by digestion ofwhole antibody with the enzyme papain to yield a light chain and aportion of the heavy chain;(2) (Fab′)₂, the fragment of the antibody that can be obtained bytreating whole antibody with the enzyme pepsin without subsequentreduction; F(ab′2) is a dimer of two Fab fragments held together by twodisulfide bonds;(3) Fv, defined as a genetically engineered fragment containing thevariable region of the light chain and the variable region of the heavychain expressed as two chains; and(4) Single chain antibody (SCA), defined as a genetically engineeredmolecule containing the variable region of the light chain and thevariable region of the heavy chain linked by a suitable polypeptidelinker as a genetically fused single chain molecule, including a scFv.

CDR grafting may be performed to alter certain properties of theantibody molecule including affinity or specificity. A non-limitingexample of CDR grafting is disclosed in U.S. Pat. No. 5,225,539.

Single-domain antibodies are isolated from the unique heavy-chainantibodies of immunized Camelidae, including camels and llamas. Thesmall antibodies are very robust and bind the antigen with high affinityin a monomeric state. U.S. Pat. No. 6,838,254 describes the productionof antibodies or fragments thereof derived from heavy chainimmunoglobulins of Camelidae.

A monoclonal antibody (mAb) is a substantially homogeneous population ofantibodies to a specific antigen. Monoclonal antibodies (mAbs) areobtained by methods known to those skilled in the art. See, for exampleKohler et al (1975); U.S. Pat. No. 4,376,110; Ausubel et al (1987-1999);Harlow et al (1988); and Colligan et al (1993), the contents of whichare incorporated entirely herein by reference. The mAbs of the presentinvention may be of any immunoglobulin class including IgG, IgM, IgE,IgA, and any subclass thereof. A hybridoma producing a mAb may becultivated in vitro or in vivo. High titers of mAbs are obtained in vivofor example wherein cells from the individual hybridomas are injectedintraperitoneally into pristine-primed Balb/c mice to produce ascitesfluid containing high concentrations of the desired mAbs. mAbs ofisotype IgM or IgG may be purified from such ascites fluid, or fromculture supernatants, using column chromatography methods well known tothose of skill in the art.

By “specific binding affinity” is meant that the antibody binds to anENDO180 polypeptide or fragment thereof with greater affinity than itbinds to another polypeptide under similar conditions.

The term “epitope” is meant to refer to that portion of a moleculecapable of being bound by an antibody which can also be recognized bythat antibody. An “antigen” is a molecule or a portion of a moleculecapable of being bound by an antibody which is additionally capable ofinducing an animal to produce antibody capable of binding to an epitopeof that antigen. An antigen may have one or more than one epitope. Thespecific reaction referred to above is meant to indicate that theantigen will react, in a highly selective manner, with its correspondingantibody and not with the multitude of other antibodies which may beevoked by other antigens.

Epitopes or antigenic determinants usually consist of chemically activesurface groupings of molecules such as amino acids or sugar side chainsand have specific three-dimensional structural characteristics as wellas specific charge characteristics.

In one embodiment, the antibody is a monoclonal antibody. In oneembodiment, the monoclonal antibody is an IgG, IgM, IgD, IgA, or IgEmonoclonal antibody. IgG subclasses are also well known to those in theart and include but are not limited to human IgG1, IgG2, IgG3 and IgG4.In one embodiment the monoclonal antibody is and IgG monoclonalantibody. In one embodiment, the monoclonal antibody is a human,humanized, or chimeric, antibody. In one embodiment, the portion of theantibody is a Fab fragment of the antibody. In one embodiment, theportion of the antibody comprises the variable domain of the antibody.In one embodiment, the portion of the antibody comprises a CDR portionof the antibody. In other embodiments the antibody is a scFv molecule.The antibodies of the present invention may be produced recombinantly(see generally Marshak et al., 1996 “Strategies for Protein Purificationand Characterization. A laboratory course manual.” Plainview, N.Y.: ColdSpring Harbor Laboratory Press, 1996) and analogs may be produced bypost-translational processing. Differences in glycosylation can providepolypeptide analogs.

The antibody may be a human or nonhuman antibody. A nonhuman antibodymay be humanized by recombinant methods to reduce its immunogenicity inman. Methods for humanizing antibodies are known to those skilled in theart.

This application provides humanized forms of the above antibodies. Asused herein, “humanized” describes antibodies wherein some, most or allof the amino acids outside the CDR regions are replaced withcorresponding amino acids derived from human immunoglobulin molecules,e.g. the human framework regions replace the non-human regions. In oneembodiment of the humanized forms of the antibodies, some, most or allof the amino acids outside the CDR regions have been replaced with aminoacids from human immunoglobulin molecules but where some, most or allamino acids within one or more CDR regions remain unchanged. Smalladditions, deletions, insertions, substitutions or modifications ofamino acids are permissible as long as they would not abrogate theability of the antibody to bind the antigen, ENDO180.

A “humanized” antibody would retain a similar antigenic specificity asthe original antibody, i.e. the ability to bind ENDO180, specificallyhuman ENDO180 receptor and would similarly be internalized by thereceptor.

One skilled in the art would know how to produce the humanizedantibodies of the subject invention. Various publications, several ofwhich are hereby incorporated by reference into this application,describe how to make humanized antibodies.

For example, the methods described in U.S. Pat. Nos. 4,816,567 and6,331,415 comprise the production of chimeric antibodies having avariable region of one antibody and a constant region of anotherantibody.

U.S. Pat. No. 5,225,539; U.S. Pat. No. 6,548,640 and U.S. Pat. No.6,982,321 describes the use of recombinant DNA technology to produce ahumanized antibody wherein the CDRs of one immunoglobulin are replacedwith the CDRs from an immunoglobulin with a different specificity suchthat the humanized antibody would recognize the target antigen but wouldnot illicit an immune response. Specifically, site directed mutagenesisis used to introduce the CDRs onto the framework region.

Other approaches for humanizing an antibody are described in WO 90/07861and corresponding patents including U.S. Pat. Nos. 5,585,089; 5,693,761;6,180,370 and 7,022,500. These patents describe a method to increase theaffinity of an antibody for the desired antigen by combining the CDRs ofa mouse monoclonal antibody with human immunoglobulin framework andconstant regions. Human framework regions can be chosen to maximizehomology with the mouse sequence. Computer modeling can be used toidentify amino acids in the framework region which are likely tointeract with the CDRs or the specific antigen and then mouse aminoacids can be used at these positions to create the humanized antibody.

The above methods are merely illustrative of some of the methods thatone skilled in the art could employ to make humanized antibodies.

The monoclonal antibody E3-8D8 represents a suitable anti-ENDO180antibody for use in the methods of the present invention. The hybridomacell E3-8D8 was deposited with the Belgian Co-ordinated Collections ofMicro-Organisms (BCCM), under the terms of the Budapest Treaty and givenAccession Number LMBP 7203CB.

Epitope Mapping

Epitope mapping studies identify the residues that are important forantibody binding. Various methods are known in the art for epitopemapping and are readily performed by one skilled in the art. Certainmethods are described in Epitope Mapping: A Practical Approach (0. M. R.Westwood, F. C. Hay; Oxford University Press, 2000), incorporated hereinby reference.

One example of an epitope mapping techniques is Synthetic LabeledPeptides Epitope Mapping whereby a set of overlapping synthetic peptidesis synthesized, each corresponding to a small segment of the linearsequence of the protein antigen, i.e. extracellular domain of ENDO180,and arrayed on a solid phase. The panel of peptides is then probed withthe test antibody, and bound antibody is detected using anenzyme-labeled secondary antibody.

Other techniques include fragmentation or cleavage and gel separation ofthe protein antigen, transfer to a membrane, probing by test antibodyand bound antibody is detected using an enzyme-labeled secondaryantibody.

Antibody Drug Development

In general monoclonal antibodies need to be designed to preserve bindingproperties (selectivity, internalization etc) and to reduce an immuneresponse in the recipient.

Specifically, the monoclonal antibody secreted from hybridoma 3E-8D8 maybe optimized for human therapeutics by one of several methods known tothose with skill in the art. In one method the variable heavy chain(V_(H)) and variable light chain (V_(L)) of the monoclonal antibody aresequenced. Once the amino acid sequence is known, the complementaritydetermining regions (CDR), heavy chain and light chain CDR3 areidentified and degenerate oligonucleotides are used to clone syntheticCDR3 into a vector to produce a recombinant vector or construct. Theconstruct may be for example a Fab fragment, a F(ab′)2 fragment, a Fvfragment, a single chain fragment or a full IgG molecule. Theconstruct(s) is expressed and a polypeptide is isolated. In someembodiments the monoclonal antibody may be further optimized bymutagenesis optimized by site directed mutagenesis to generate a CDR3domain having substantial identity to the original CDR3.

Therapeutic Agents

The therapeutic agent or active agents according to the presentinvention includes nucleotide and non-nucleotide agents, includingoligonucleotides such as antisense (AS), miRNA and unmodified andchemically modified siRNA compounds. A preferred therapeutic agent is asiRNA compound.

In some embodiments the siRNA targets and reduces expression of a targetgene by RNA interference.

The therapeutic oligonucleotides of the present invention aresynthesized by any method known in the art for ribonucleic ordeoxyribonucleic nucleotides. For example, a commercial polynucleotidesynthesizer (e.g. Applied Biosystems 380B DNA synthesizer) can be used.When fragments are used, two or more such sequences can be synthesizedand linked together for use in the present invention. Although a siRNAis the preferred therapeutic agent according to the present invention,the present invention encompasses a conjugate or mixture wherein thetherapeutic agent is selected from alkylating agents such as thiotepaand CYTOXAN® cyclosphosphamide; alkyl sulfonates such as busulfan,improsulfan and piposulfan; aziridines such as benzodopa, carboquone,meturedopa, and uredopa; ethylenimines and methylamelamines includingaltretamine, triethylenemelamine, trietylenephosphoramide,triethiylenethiophosphoramide and trimethylolomelamine; acetogenins(especially bullatacin and bullatacinone); delta-9-tetrahydrocannabinol(dronabinol, MARINOL®); beta-lapachone; lapachol; colchicines; betulinicacid; a camptothecin (including the synthetic analog topotecan(HYCAMTIN®), CPT-11 (irinotecan, CAMPTOSAR®), acetylcamptothecin,scopolectin, and 9-aminocamptothecin); bryostatin; callystatin; CC-1065(including its adozelesin, carzelesin and bizelesin synthetic analogs);podophyllotoxin; podophyllinic acid; teniposide; cryptophycins(particularly cryptophycin 1 and cryptophycin 8); dolastatin;duocarmycin (including the synthetic analogs, KW-2189 and CB1-TM1);eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogenmustards such as chlorambucil, chlornaphazine, cholophosphamide,estramustine, ifosfamide, mechlorethamine, mechlorethamine oxidehydrochloride, melphalan, novembichin, phenesterine, prednimustine,trofosfamide, uracil mustard; nitrosureas such as carmustine,chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine;antibiotics such as the enediyne antibiotics (e.g., calicheamicin,especially calicheamicin gamma1I and calicheamicin omegaI1 (see, e.g.,Agnew, Chem. Intl. Ed. Engl., 1994. 33: 183-186); dynemicin, includingdynemicin A; an esperamicin; as well as neocarzinostatin chromophore andrelated chromoprotein enediyne antibiotic chromophores), aclacinomysins,actinomycin, authramycin, azaserine, bleomycins, cactinomycin,carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin,daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin(including ADRIAMYCIN®, morpholino-doxorubicin,cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, doxorubicin HClliposome injection (DOXIL®) and deoxydoxorubicin), epirubicin,esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C,mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin,puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin,tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such asmethotrexate, gemcitabine (GEMZAR®), tegafur (UFTORAL®), capecitabine(XELODA®), an epothilone, and 5-fluorouracil (5-FU); folic acid analogssuch as denopterin, methotrexate, pteropterin, trimetrexate; purineanalogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine;pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine,carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine,floxuridine; androgens such as calusterone, dromostanolone propionate,epitiostanol, mepitiostane, testolactone; anti-adrenals such asaminoglutethimide, mitotane, trilostane; folic acid replenisher such asfrolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinicacid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate;defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate;etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine;maytansinoids such as maytansine and ansamitocins; mitoguazone;mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet;pirarubicin; losoxantrone; 2-ethylhydrazide; procarbazine; PSK®polysaccharide complex; razoxane; rhizoxin; sizofiran; spirogermanium;tenuazonic acid; triaziquone; 2,2′,2″-trichlorotriethylamine;trichothecenes (especially T-2 toxin, verracurin A, roridin A andanguidine); urethan; vindesine (ELDISINE®, FILDESIN®); dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); thiotepa; taxoids, e.g., paclitaxel (TAXOL®),albumin-engineered nanoparticle formulation of paclitaxel (ABRAXANE™),and doxetaxel (TAXOTERE®); chloranbucil; 6-thioguanine; mercaptopurine;methotrexate; a platinum analog such as cisplatin and carboplatin;vinblastine (VELBAN®); platinum; etoposide (VP-16); ifosfamide;mitoxantrone; vincristine (ONCOVIN®); oxaliplatin; leucovovin;vinorelbine (NAVELBINE®); novantrone; edatrexate; daunomycin;aminopterin; ibandronate; topoisomerase inhibitor RFS 2000;difluoromethylornithine (DMFO); a retinoid such as retinoic acid;pharmaceutically acceptable salts, acids or derivatives of any of theabove; as well as combinations of two or more of the above such as CHOP,an abbreviation for a combined therapy of cyclophosphamide, doxorubicin,vincristine, and prednisolone, and FOLFOX, an abbreviation for atreatment regimen with oxaliplatin (ELOXATIN®) combined with 5-FU andleucovovin.

Also included in this definition are anti-hormonal agents that act toregulate, reduce, block, or inhibit the effects of hormones that canpromote the growth of cancer, and are often administered as systemic, orwhole-body treatment. They may be hormones themselves. Examples includeanti-estrogens and selective estrogen receptor modulators (SERMs),including, for example, tamoxifen (including NOLVADEX® tamoxifen),raloxifene (EVISTA®), droloxifene, 4-hydroxytamoxifen, trioxifene,keoxifene, LY117018, onapristone, and toremifene (FARESTON®);anti-progesterones; estrogen receptor down-regulators (ERDs); agentsthat function to suppress or shut down the ovaries, for example,leutinizing hormone-releasing hormone (LHRH) agonists such as leuprolideacetate (LUPRON® and ELIGARD®), goserelin acetate, buserelin acetate andtripterelin; other anti-androgens such as flutamide, nilutamide andbicalutamide; and aromatase inhibitors such as, for example,4(5)-imidazoles, aminoglutethimide, megestrol acetate (MEGASE®),exemestane (AROMASIN®), formestanie, fadrozole, vorozole (RIVISOR®),letrozole (FEMARA®), and anastrozole (ARIMIDEX®). In addition,bisphosphonates such as clodronate (for example, BONEFOS® or OSTAC®),etidronate (DIDROCAL®), NE-58095, zoledronic acid/zoledronate (ZOMETA®),alendronate (FOSAMAX®), pamidronate (AREDIA®), tiludronate (SKELID®), orrisedronate (ACTONEL®); as well as troxacitabine (a 1,3-dioxolanenucleoside cytosine analog); siRNA, ribozyme and antisenseoligonucleotides, particularly those that inhibit expression of genes insignaling pathways implicated in aberrant cell proliferation; vaccinessuch as THERATOPE® vaccine and gene therapy vaccines, for example,ALLOVECTIN® vaccine, LEUVECTIN® vaccine, and VAXID® vaccine;topoisomerase 1 inhibitor (e.g., LURTOTECAN®); rmRH (e.g., ABARELIX®);lapatinib ditosylate (an ErbB-2 and EGFR dual tyrosine kinasesmall-molecule inhibitor also known as GW572016); COX-2 inhibitors suchas celecoxib (CELEBREX®;4-(5-(4-methylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl)benzenesulfonamide; and pharmaceutically acceptable salts, acids orderivatives of any of the above.

As used herein, the term “polypeptide” refers to, in addition to apolypeptide, a peptide and a full protein and includes isolated andrecombinant polypeptides. As used herein, “biological function” refersto the biological property of the molecule and in this context means anin vivo effector or antigenic function or activity that is directly orindirectly performed by a naturally occurring polypeptide or nucleicacid molecule. Biological functions include but are not limited toreceptor binding, any enzymatic activity or enzyme modulatory activity,any carrier binding activity, any hormonal activity, any activity ininternalizing molecules or translocation from one compartment toanother, any activity in promoting or inhibiting adhesion of cells toextracellular matrix or cell surface molecules, or any structural role,as well as having the nucleic acid sequence encode functional proteinand be expressible. The antigenic functions essentially mean thepossession of an epitope or an antigenic site that is capable ofcross-reacting with antibodies raised against a naturally occurringprotein. Biologically active analogs share an effector function of thenative polypeptide that may, but need not, in addition possess anantigenic function.

Measurement of the level of the ENDO180 polypeptide may be determined bya method selected from the group consisting of immunohistochemistry,western blotting, ELISA, antibody microarray hybridization and targetedmolecular imaging. Such methods are well-known in the art, for exampleimmunohistochemistry, western blotting, antibody microarrayhybridization, and targeted molecular imaging.

Measurement of the level of ENDO180 polynucleotide may be determined bya method selected for example from: RT-PCR analysis, in-situhybridization, polynucleotide microarray and Northern blotting. Suchmethods are well known in the art.

Antisense Molecules

In some embodiments the therapeutic agent is an antisenseoligonucleotide. By the term “antisense” (AS) or “antisense fragment” ismeant a polynucleotide fragment (comprising either deoxyribonucleotides,ribonucleotides or a mixture of both) having inhibitory antisenseactivity, said activity causing a decrease in the expression of theendogenous genomic copy of the corresponding gene. An AS polynucleotideis a polynucleotide which comprises consecutive nucleotides having asequence of sufficient length and homology to a sequence present withinthe sequence of the target gene to permit hybridization of the AS to thegene. Many reviews have covered the main aspects of antisense (AS)technology and its therapeutic potential (Aboul-Fadl T., Curr Med Chem.2005, 12(19):2193-214; Crooke S T, Curr Mol Med. 2004, 4(5):465-87;Crooke S T, Ann Rev Med. 2004, 55:61-95; Vacek M et al, Cell Mol LifeSci. 2003, 60(5):825-33; Cho-Chung Y S, Arch Pharm Res. 2003, 26(3):183-91. There are further reviews on the chemical (Crooke et al.,Hematol Pathol. 1995, 9(2):59-72), cellular (Wagner, Nature. 1994,372(6504):333-5) and therapeutic (Scanlon, et al, FASEB J. 1995, 9(13):1288-96) aspects of AS technology. Antisense intervention in theexpression of specific genes can be achieved by the use of modified ASoligonucleotide sequences (for recent reports see Lefebvre-d'Hellencourtet al, 1995; Agrawal, 1996; LevLehman et al, 1997).

AS oligonucleotide sequences may be short sequences of DNA, typically15-30 mer but may be as small as 7-mer (Wagner et al, Nat. Biotech.1996, 14(7):840-4), designed to complement a target mRNA of interest andform an RNA:AS duplex. This duplex formation can prevent processing,splicing, transport or translation of the relevant mRNA.

Moreover, certain AS nucleotide sequences can elicit cellular RNase Hactivity when hybridized with their target mRNA, resulting in mRNAdegradation (Calabretta et al, Semin Oncol. 1996, 23(1):78-87). In thatcase, RNaseH will cleave the RNA component of the duplex and canpotentially release the AS to further hybridize with additionalmolecules of the target RNA. An additional mode of action results fromthe interaction of AS with genomic DNA to form a triple helix, which canbe transcriptionally inactive.

The sequence target segment for the antisense oligonucleotide isselected such that the sequence exhibits suitable energy relatedcharacteristics important for oligonucleotide duplex formation withtheir complementary templates, and shows a low potential forself-dimerization or self-complementation (Anazodo et al, 1996, BBRC.229:305-309). For example, the computer program OLIGO (Primer AnalysisSoftware, Version 3.4), can be used to determine antisense sequencemelting temperature, free energy properties, and to estimate potentialself-dimer formation and self-complimentary properties. The programallows the determination of a qualitative estimation of these twoparameters (potential self-dimer formation and self-complimentary) andprovides an indication of “no potential” or “some potential” or“essentially complete potential”. Using this program target segments aregenerally selected that have estimates of no potential in theseparameters. However, segments can be used that have “some potential” inone of the categories. A balance of the parameters is used in theselection as is known in the art. Further, the oligonucleotides are alsoselected as needed so that analog substitution does not substantiallyaffect function.

Phosphorothioate antisense oligonucleotides do not normally showsignificant toxicity at concentrations that are effective and exhibitsufficient pharmacodynamic half-lives in animals (Agrawal, et al., PNASUSA. 1997, 94(6):2620-5) and are nuclease resistant. Antisenseoligonucleotide inhibition of basic fibroblast growth factor (bFGF),having mitogenic and angiogenic properties, suppressed 80% of growth inglioma cells (Morrison, J Biol Chem. 1991 266(2):728-34) in a saturableand specific manner. Being hydrophobic, antisense oligonucleotidesinteract well with phospholipid membranes (Akhter et al., NAR. 1991,19:5551-5559). Following their interaction with the cellular plasmamembrane, they are actively (or passively) transported into living cells(Loke et al., PNAS 1989, 86(10):3474-8), in a saturable mechanismpredicted to involve specific receptors (Yakubov et al., PNAS, 198986(17):6454-58)

Ribozymes

A “ribozyme” is an RNA molecule that possesses RNA catalytic ability(see Cech for review) and cleaves a specific site in a target RNA. Inaccordance with the present invention, ribozymes which cleave mRNA maybe utilized as a therapeutic agent. This may be necessary in cases whereantisense therapy is limited by stoichiometric considerations (Sarver etal., 1990, Gene Regulation and Aids, pp. 305-325). Ribozymes can then beused that will target the a gene associated with a bone marrow disease.The number of RNA molecules that are cleaved by a ribozyme is greaterthan the number predicted by stoichiometry. (Hampel and Tritz, Biochem.1989, 28(12):4929-33; Uhlenbeck, Nature. 1987.328(6131):596-600).Ribozymes catalyze the phosphodiester bond cleavage of RNA. Severalribozyme structural families have been identified including Group Initrons, RNase P, the hepatitis delta virus ribozyme, hammerheadribozymes and the hairpin ribozyme originally derived from the negativestrand of the tobacco ringspot virus satellite RNA (sTRSV) (U.S. Pat.No. 5,225,347). The latter two families are derived from viroids andvirusoids, in which the ribozyme is believed to separate monomers fromoligomers created during rolling circle replication (Symons, 1989 and1992). Hammerhead and hairpin ribozyme motifs are most commonly adaptedfor trans-cleavage of mRNAs for gene therapy (Sullivan, 1994). Ingeneral the ribozyme has a length of from about 30-100 nucleotides.Delivery of ribozymes is similar to that of AS fragments and/or siRNAmolecules.

siRNA and RNA Interference

RNA interference (RNAi) is a phenomenon involving double-stranded (ds)RNA-dependent gene-specific posttranscriptional silencing. Initialattempts to study this phenomenon and to manipulate mammalian cellsexperimentally were frustrated by an active, non-specific antiviraldefense mechanism which was activated in response to long dsRNAmolecules (Gil et al., Apoptosis, 2000. 5:107-114). Later, it wasdiscovered that synthetic duplexes of 21 nucleotide RNAs could mediategene specific RNAi in mammalian cells, without stimulating the genericantiviral defense mechanisms Elbashir et al. Nature 2001, 411:494-498and Caplen et al. PNAS 2001, 98:9742-9747). As a result, smallinterfering RNAs (siRNAs), which are short double-stranded RNAs, havebeen widely used to inhibit gene expression and understand genefunction.

RNA interference (RNAi) is mediated by small interfering RNAs (siRNAs)(Fire et al, Nature 1998, 391:806) or microRNAs (miRNAs) (Ambros V.Nature 2004, 431:350-355); and Bartel D P. Cell. 2004 116(2):281-97).The corresponding process is commonly referred to as specificpost-transcriptional gene silencing when observed in plants and asquelling when observed in fungi.

A siRNA is a double-stranded RNA which down-regulates or silences (i.e.fully or partially inhibits) the expression of an endogenous orexogenous gene/mRNA. RNA interference is based on the ability of certaindsRNA species to enter a specific protein complex, where they are thentargeted to complementary cellular RNA (i.e. mRNA), which theyspecifically degrade or cleave. Thus, the RNA interference responsefeatures an endonuclease complex containing siRNA, commonly referred toas an RNA-induced silencing complex (RISC), which mediates cleavage ofsingle-stranded RNA having a sequence complementary to the antisensestrand of the siRNA duplex. Cleavage of the target RNA may take place inthe middle of the region complementary to the antisense strand of thesiRNA duplex (Elbashir, et al., Genes Dev., 2001, 15:188). In moredetail, longer dsRNAs are digested into short (17-29 bp) dsRNA fragments(also referred to as short inhibitory RNAs or “siRNAs”) by type IIIRNAses (DICER, DROSHA, etc., (see Bernstein et al., Nature, 2001,409:363-6 and Lee et al., Nature, 2003, 425:415-9). The RISC proteincomplex recognizes these fragments and complementary mRNA. The wholeprocess is culminated by endonuclease cleavage of target mRNA (McManusand Sharp, Nature Rev Genet, 2002, 3:737-47; Paddison and Hannon, CurrOpin Mol Ther. 2003, 5(3): 217-24). (For additional information on theseterms and proposed mechanisms, see for example, Bernstein, et al., RNA.2001, 7(11):1509-21; Nishikura, Cell. 2001, 107(4):415-8 and PCTPublication No. WO 01/36646).

Studies have revealed that siRNA can be effective in vivo in mammalsincluding humans. Specifically, Bitko et al., showed that specificsiRNAs directed against the respiratory syncytial virus (RSV)nucleocapsid N gene are effective in treating mice when administeredintranasally (Nat. Med. 2005, 11(1):50-55). For reviews of therapeuticapplications of siRNAs see for example Batik (Mol. Med 2005, 83:764-773) and Chakraborty (Current Drug Targets 2007 8(3):469-82). Inaddition, clinical studies with short siRNAs that target the VEGFR1receptor in order to treat age-related macular degeneration (AMD) havebeen conducted in human patients (Kaiser, Am J Ophthalmol. 2006142(4):660-8). Further information on the use of siRNA as therapeuticagents may be found in Durcan, 2008. Mol. Pharma. 5(4):559-566; Kim andRossi, 2008. BioTechniques 44:613-616; Grimm and Kay, 2007, JCI,117(12):3633-41.

The siRNA according to the invention is unmodified, recombinant orchemically modified. Examples of chemical modifications useful insynthesizing siRNA are disclosed in PCT Patent Publication No. WO2009/044392, assigned to the assignee of the present invention, andhereby incorporated by reference in its entirety.

Pharmaceutical Compositions

The present invention provides for a pharmaceutical compositioncomprising any one of the above compounds and a pharmaceuticallyacceptable excipient. In some embodiments the pharmaceutical compositionaccording to the present invention comprises one of an anti-ENDO180antibody or antigen-binding fragment thereof selected from

-   -   a) the monoclonal antibody produced by the hybridoma cell line        E3-8D8 (BCCM Accession Number LMBP 7203CB);    -   b) an antibody or fragment thereof that binds to the same        epitope as the antibody of (a);    -   c) a humanized antibody of (a) or (b);    -   d) a recombinant polypeptide comprising amino acid sequences set        forth in SEQ ID NO: 6 or a variant thereof; and    -   e) a recombinant polypeptide comprising CDR3 having amino acid        sequences set forth in SEQ ID NO:7 and 8, or variants thereof;    -   f) a conjugate of any one of the above (a)-(e) conjugated to a        moiety;        wherein upon contact with a cell expressing ENDO180 the antibody        or antigen binding fragment thereof is internalized into the        cell; and        a pharmaceutically acceptable vehicle or carrier.

In some embodiments the carrier comprises a lipid particle or alipidated glycosaminoglycan.

In another aspect the invention provides compounds including a) ananti-ENDO180 antibody or antigen binding fragment thereof; b) a moietyselected from a detectable label, a cytotoxic agent or a therapeuticagent, and c) a nanocarrier.

In various embodiments the nanocarrier is a polysaccharide-basednanoparticle. In various embodiments, tripartite compounds of theinvention can be represented by one of the formulas:

A-X-Y

X-A-Y or

X Y A

wherein A represents a detectable label, a cytotoxic agent or atherapeutic agent;X represents a nanocarrier; andY represents an anti-ENDO180 antibody or antigen-binding fragmentthereof.

The disclosed compounds are designed to target particular cells ortissues expressing the ENDO180 polypeptide, so that a detectable label,a cytotoxic agent or a therapeutic agent is delivered to the desiredcell more effectively and with high specificity. For example, oneembodiment of the disclosure includes compounds that target cancerouscell and/or tissues.

As such, certain examples of these compounds include a an anti-ENDO180antibody or antigen binding fragment thereof that binds to an ENDO180receptor that is present in a higher concentration on a cancer cell thanon a normal cell. Embodiments of the disclosed compounds exploit theup-regulated expression of ENDO180 receptors in diseased cells andtissue to selectively deliver a therapeutic agent to such a cell.

In various embodiments the nanocarrier is a polysaccharide-basednanoparticle. In certain embodiments the polysaccharide is aglycosaminoglycan or a mucopolysaccharide. In various embodiments theglycosaminoglycan is selected from the group comprising, without beinglimited to, hyaluronic acid, chondroitin sulfate, dermatan sulfate,keratan sulfate, heparin, heparan sulfate and combinations thereof.

In certain embodiments the nanocarrier includes organic polymers,including, without being limited to, organic polymers that self assembleto form a self-assembled nanoparticle, which provides an effectivelymultivalent species. In such embodiments the self-assemblednanoparticles can include the same or different compounds. For example,the self-assembled nanoparticles include compounds having ananti-ENDO180 antibody or antigen-binding fragment thereof a moietyselected from a detectable label, a cytotoxic agent or a therapeuticagent; and the nanocarrier components.

Embodiments of the disclosed compounds include a plurality oftherapeutic agents, detectable labels of cytotoxic agents. In suchembodiments, the compounds include different therapeutic agents, imagingagents and cytotoxic agents. In certain embodiments compounds having twoor more therapeutic agents have increased therapeutic efficiency due tomultivalent effects

The present invention further provides for a pharmaceutical compositioncomprising the disclosed compounds and conjugates, formulated foradministration to a subject. An additional aspect of the presentinvention provides for methods of treating a subject having aproliferative disease including cancer, metastatic disease and fibrosis,using the disclosed compounds, and hence pharmaceutical compositions areprovided herein for this purpose.

In preferred embodiments the therapeutic agent is a chemically modifiedsiRNA compound that inhibits expression of a target gene set forth inTable A.

In some embodiments the compositions comprise a mixture of two or moredifferent therapeutic agents including two or mores siRNA that target asingle gene or multiple genes.

The invention further provides a pharmaceutical composition comprisingat least one compound of the invention covalently or non-covalentlybound to one or more compounds of the invention in an amount effectiveto inhibit target gene expression or activity; and a pharmaceuticallyacceptable carrier.

The pharmaceutically “effective amount” for purposes herein is thusdetermined by such considerations as are known in the art. The amountmust be effective to achieve improvement including but not limited toimproved survival rate or more rapid recovery, or improvement orelimination of symptoms and other indicators as are selected asappropriate measures by those skilled in the art. The compounds of thepresent invention can be administered by any of the conventional routesof administration. It should be noted that the compound can beadministered as the compound or as pharmaceutically acceptable salt andcan be administered alone or as an active ingredient in combination withpharmaceutically acceptable carriers, solvents, diluents, excipients,adjuvants and vehicles. The compounds can be administered orally,subcutaneously or parenterally including intravenous, intraarterial,intramuscular, intraperitoneally, and intranasal administration as wellas intrathecal and infusion techniques. Implants of the compounds arealso useful. Liquid forms may be prepared for injection, the termincluding subcutaneous, transdermal, intravenous, intramuscular,intrathecal, and other parental routes of administration. The liquidcompositions include aqueous solutions, with and without organiccosolvents, aqueous or oil suspensions, emulsions with edible oils, aswell as similar pharmaceutical vehicles. In addition, under certaincircumstances the compositions for use in the novel treatments of thepresent invention may be formed as aerosols, for intranasal and likeadministration. The patient being treated is a warm-blooded animal and,in particular, mammals including man. The pharmaceutically acceptablecarriers, solvents, diluents, excipients, adjuvants and vehicles as wellas implant carriers generally refer to inert, non-toxic solid or liquidfillers, diluents or encapsulating material not reacting with the activeingredients of the invention and they include liposomes, lipidatedglycosaminoglycans and microspheres. Examples of delivery systems usefulin the present invention include U.S. Pat. Nos. 5,225,182; 5,169,383;5,167,616; 4,959,217; 4,925,678; 4,487,603; 4,486,194; 4,447,233;4,447,224; 4,439,196; and 4,475,196. Many other such implants, deliverysystems, and modules are well known to those skilled in the art.

In general, the active dose of compound for humans is in the range offrom 1 ng/kg to about 20-100 mg/kg body weight per day, preferably about0.01 mg to about 2-10 mg/kg body weight per day, in a regimen of onedose per day or twice or three or more times per day for a period of 1-2weeks or longer, preferably for 24- to 48 hrs or by continuous infusionduring a period of 1-2 weeks or longer.

Additionally, the invention provides a method of inhibiting theexpression of the genes of the present invention by at least 50% ascompared to a control comprising contacting an mRNA transcript of thegene of the present invention with one or more of the compounds of theinvention.

In one embodiment the therapeutic agent inhibits a target gene, wherebythe inhibition is selected from the group comprising inhibition of genefunction, inhibition of polypeptide and inhibition of mRNA expression.

The pharmaceutical composition is formulated to provide for a singledosage administration or a multi-dosage administration.

In various embodiments the pharmaceutical composition comprising aconjugate or mixture of the invention is administered intravenously,intramuscularly, locally, or subcutaneously to the subject.

The pharmaceutical composition according to the present invention canalso be used in a method for preventing and/or treating a disease asdisclosed herein, whereby the method comprises the administration of aconjugate according to the present invention, a mixture according to thepresent invention or a pharmaceutical composition or medicamentaccording to the present invention for any of the diseases describedherein.

Diagnostics

The compounds of the invention are useful in diagnosing ENDO180expressing cells in biological samples.

Delivery

In some embodiments the antibodies, antigen-binding fragments and/orconjugates of the present invention are delivered to the target tissueby direct application of the naked molecules prepared with a carrier ora diluent.

The term “naked molecule” refers to antibodies, antigen-bindingfragments or conjugates that are free from any delivery vehicle thatacts to assist, promote or facilitate entry into the cell, includingviral sequences, viral particles, lipid particles, liposomeformulations, lipofectin or precipitating agents and the like. Forexample, siRNA in PBS is “naked siRNA”. However, in some embodiments theantibodies, antigen-binding fragments or conjugates of the invention aredelivered with lipid particles, polysaccharide particles or combinationsthereof, liposome formulations, or lipofectin formulations and the likeand can be prepared by methods well known to those skilled in the art.Such methods are described, for example, in U.S. Pat. Nos. 5,593,972,5,589,466, and 5,580,859, which are herein incorporated by reference.

In other embodiments the antibodies, antigen-binding fragments orconjugates of the invention are attached to or are entrapped within adelivery particle. In some embodiments the ENDO180 binding domain of theconjugate molecule is exposed on the external surface of deliveryparticle. In some embodiments the delivery particle is a liposome. Inspecific preferred embodiment the delivery particle is a lipidatedglycosaminoglycan particle. Such particles are described, for example inU.S. patent application Ser. No. 10/487,022 (Publication No.20040241248), U.S. patent application Ser. No. 11/718,485 (PublicationNo. 20080248092), U.S. patent application Ser. No. 11/632,647(Publication No. 20090022656), which are herein incorporated byreference Without wishing to be bound to theory, the antibody orantigen-binding fragment thereof is exposed on the surface of thedelivery particle and homes in on or targets the target cell expressingan ENDO180 polypeptide on its surface.

The pharmaceutically acceptable carriers, solvents, diluents,excipients, adjuvants and vehicles as well as implant carriers generallyrefer to inert, non-toxic solid or liquid fillers, diluents orencapsulating material not reacting with the active ingredients of theinvention and they include liposomes and microspheres. Examples ofdelivery systems useful in the present invention include U.S. Pat. Nos.5,225,182; 5,169,383; 5,167,616; 4,959,217; 4,925,678; 4,487,603;4,486,194; 4,447,233; 4,447,224; 4,439,196; and 4,475,196. Many othersuch implants, delivery systems, and modules are well known to thoseskilled in the art. In one specific embodiment of this invention topicaland transdermal formulations may be selected. The siRNAs orpharmaceutical compositions of the present invention are administeredand dosed in accordance with good medical practice, taking into accountthe clinical condition of the individual patient, the disease to betreated, the site and method of administration, scheduling ofadministration, patient age, sex, body weight and other factors known tomedical practitioners.

The “therapeutically effective dose” for purposes herein is thusdetermined by such considerations as are known in the art. The dose mustbe effective to achieve improvement including but not limited toimproved survival rate or more rapid recovery, or improvement orelimination of symptoms and other indicators as are selected asappropriate measures by those skilled in the art.

In general, the active dose of compound for humans is in the range offrom 1 ng/kg to about 20-100 mg/kg body weight per day, preferably about0.01 mg to about 2-10 mg/kg body weight per day, in a regimen of onedose per day or twice or three or more times per day for a period of 1-4weeks or longer.

The compounds of the present invention can be administered by any of theconventional routes of administration. It should be noted that thecompound can be administered as the compound or as pharmaceuticallyacceptable salt and can be administered alone or as an active ingredientin combination with pharmaceutically acceptable carriers, solvents,diluents, excipients, adjuvants and vehicles. The compounds can beadministered orally, subcutaneously or parenterally includingintravenous, intraarterial, intramuscular, intraperitoneally, andintranasal, inhalation, transtympanic administration as well asintrathecal and infusion techniques. Implants of the compounds are alsouseful. Liquid forms may be prepared for injection, the term includingsubcutaneous, transdermal, intravenous, intramuscular, intrathecal,intranasal and other parental routes of administration. The liquidcompositions include aqueous solutions, with and without organicco-solvents, aqueous or oil suspensions, emulsions with edible oils, aswell as similar pharmaceutical vehicles. In a particular embodiment, theadministration comprises intravenous administration. In anotherembodiment the administration comprises topical or local administration.In addition, in certain embodiments the compositions for use in thenovel treatments of the present invention may be formed as aerosols, forexample for intranasal administration.

In certain embodiments, oral compositions (such as tablets, suspensions,solutions) may be effective for local delivery to the oral cavity suchas oral composition suitable for mouthwash for the treatment of oralmucositis.

Liquid forms are prepared for drops or spray. The liquid compositionsinclude aqueous solutions, with and without organic co-solvents, aqueousor oil suspensions, emulsions with oils, as well as similarpharmaceutical vehicles. In some embodiments administration comprisestopical or local administration.

These compounds are administered to humans and other animals for therapyby any suitable route of administration to the eye, as by, for example,a spray or drops, and topically, as by ointments, suspensions or drops.

In preferred embodiments the subject being treated is a warm-bloodedanimal and, in particular, mammals including human.

Suitable methods for delivery of the compounds of present inventioninclude, among others, systemic delivery, transfection, lipofection, andelectroporation. In a further aspect the present invention is related toa pharmaceutical composition comprising a delivery molecule-therapeuticagent conjugate or an anti-ENDO180 antibody or anti-ENDO180antibody-therapeutic agent mixture according to the present inventionand, a pharmaceutically acceptable carrier, diluent or adjuvants orother vehicle(s).

Preferably, such carrier, diluents, adjuvants and vehicles are inert,and non-toxic. The pharmaceutical composition is in its variousembodiments adapted for administration in various ways. Suchadministration comprises systemic and local administration as well asoral, subcutaneous, parenteral, intravenous, intraarterial,intramuscular, intraperitoneal, intranasal, intrathecal, transtympanicand intraocular.

In some embodiments the vehicle is selected from a lipid particle, apolysaccharide particle or a combination thereof and a lipidatedglycosaminoglycan particle (gagomer). In various embodiments thedelivery molecule-therapeutic agent conjugate or antibody-therapeuticmixture is at least partially exposed on the external surface of thelipid particle or the lipidated glycosaminoglycan particle.

It will be acknowledged by the one skilled in the art that the amount ofthe pharmaceutical composition and the respective siRNA depends on theclinical condition of the individual patient, the site and method ofadministration, scheduling of administration, patient age, sex,bodyweight and other factors known to medical practitioners. Thepharmaceutically effective amount for purposes of prevention and/ortreatment is thus determined by such considerations as are known in themedical arts. Preferably, the amount is effective to achieve improvementincluding but limited to improve the diseased condition or to providefor a more rapid recovery, improvement or elimination of symptoms andother indicators as are selected as appropriate measures by thoseskilled in the medical arts.

Combination Therapy

In various embodiments the present invention relates to combinationtherapy. In one embodiment, the co-administration of two or moretherapeutic agents achieves a synergistic effect, i.e., a therapeuticaffect that is greater than the sum of the therapeutic effects of theindividual components of the combination. In another embodiment, theco-administration of two or more therapeutic agents achieves an additiveeffect.

The active ingredients that comprise a combination therapy may beadministered together via a single dosage form or by separateadministration of each active agent. In certain embodiments, the firstand second therapeutic agents are administered in a single dosage form.The agents may be formulated into a single tablet, pill, capsule, orsolution for parenteral administration and the like. Alternatively, thefirst therapeutic agent and the second therapeutic agents may beadministered as separate compositions. The first active agent may beadministered at the same time as the second active agent or the firstactive agent may be administered intermittently with the second activeagent. The length of time between administration of the first and secondtherapeutic agent may be adjusted to achieve the desired therapeuticeffect. For example, the second therapeutic agent may be administeredonly a few minutes (e.g., 1, 2, 5, 10, 30, or 60 min) or several hours(e.g., 2, 4, 6, 10, 12, 24, or 36 hr) after administration of the firsttherapeutic agent. In certain embodiments, it may be advantageous toadminister more than one dosage of one of the therapeutic agents betweenadministrations of the second therapeutic agent. For example, the secondtherapeutic agent may be administered at 2 hours and then again at 10hours following administration of the first therapeutic agent.Alternatively, it may be advantageous to administer more than one dosageof the first therapeutic agent between administrations of the secondtherapeutic agent. Importantly, it is preferred that the therapeuticeffects of each active ingredient overlap for at least a portion of theduration of each therapeutic agent so that the overall therapeuticeffect of the combination therapy is attributable in part to thecombined or synergistic effects of the combination therapy.

The present invention relates to compounds and the use of compounds,which down-regulate the expression of the genes of the inventionparticularly to conjugates comprising small interfering RNAs (siRNAs).Methods, molecules and compositions useful for inhibition of targetgenes are discussed herein at length, and any of said molecules and/orcompositions may be beneficially employed in the treatment of a subjectsuffering from a proliferative or fibrotic disease.

Methods of Treatment

An additional aspect of the present invention provides for methods oftreating a proliferative disease including cancer, metastatic diseaseand fibrosis. Methods for therapy of diseases or disorders associatedwith uncontrolled, pathological cell growth, e.g. cancer, psoriasis,autoimmune diseases, inter alia, and methods for therapy of diseasesassociated with ischemia and lack of proper blood flow, e.g. myocardialinfarction (MI) and stroke, are provided. In particular, the compoundsand compositions of the invention are useful in treating proliferativediseases in which ENDO180 is expressed in at least a portion of thediseased cells and or tissue.

“Cancer” or “Tumor” refers to an abnormal proliferation of cells. Theseterms include both primary tumors, which may be benign or malignant, aswell as secondary tumors, or metastases which have spread to other sitesin the body. Examples of proliferative diseases include, inter alia:carcinoma (e.g.: breast, colon and lung), leukemia such as B cellleukemia, lymphoma such as B-cell lymphoma, blastoma such asneuroblastoma and melanoma and sarcoma. It will be acknowledged that thepharmaceutical composition according to the present invention can beused for any disease which involves undesired development or growth ofvasculature including angiogenesis, as well as any of the diseases andconditions described herein.

The present invention provides methods and compositions for treating apatient suffering from a cancerous proliferative disease, (e.g. lungcancer, breast cancer, cervical cancer, colon cancer, gastric cancer,kidney cancer, leukemia, liver cancer, lymphoma, ovarian cancer,pancreatic cancer, prostate cancer, rectal cancer, sarcoma, skin cancer,testicular cancer, and uterine cancer) which the cancer cell expressesENDO180 polypeptide. In one particular embodiment, the cancer is renalcancer including RCC and TCC.

“Cancer and “cancerous disease” are used interchangeably and refer to adisease that is caused by or results in inappropriately high levels ofcell division, inappropriately low levels of apoptosis, or both.Examples of cancerous diseases include, without limitation, leukemias(e.g., acute leukemia, acute lymphocytic leukemia, acute myelocyticleukemia, acute myeloblastic leukemia, acute promyelocytic leukemia,acute myelomonocytic leukemia, acute monocytic leukemia, acuteerythroleukemia, chronic leukemia, chronic myelocytic leukemia, chroniclymphocytic leukemia), polycythemia vera, lymphoma (Hodgkin's disease,non-Hodgkin's disease), Waldenstrom's macroglobulinemia, heavy chaindisease, and solid tumors such as sarcomas and carcinomas (e.g.,fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenicsarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangio sarcoma,lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor,leiomyo sarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer,breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma,basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceousgland carcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, nile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, uterinecancer, testicular cancer, lung carcinoma, small cell lung carcinoma,bladder carcinoma, epithelial carcinoma, glioma, astrocytoma,medulloblastoma, crailiopharyngioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodenroglioma, schwamioma,meningioma, melanoma, neuroblastoma, and retinoblastoma). Metastases ofa primary cancer is included. In some preferred embodiments thecompounds of the present invention are useful in treating renal cancer,breast cancer, ovarian cancer and metastases thereof in various organsincluding lung and bone.

As used herein, the term “proliferative disease” refers to any diseasein which cellular proliferation, either malignant or benign, contributesto the pathology of the condition. Such unwanted proliferation is thehallmark of cancer and many chronic inflammatory diseases, thus examplesof “proliferative disease” include the cancers listed supra and chronicinflammatory proliferative diseases such as psoriasis, inflammatorybowel disease and rheumatoid arthritis; proliferative cardiovasculardiseases such as restenosis; proliferative ocular disorders such asdiabetic retinopathy; and benign hyperproliferative diseases such ashemangiomas.

Fibrotic Disease

Fibrotic diseases are a group of chronic disease characterized by theexcess production of a fibrous material called the extracellular matrix,which contributes to abnormal changes in tissue architecture andinterferes with normal organ function. Millions of people worldwidesuffer from these chronic diseases, that are often life threatening.Unfortunately, although fibrosis is widely prevalent, debilitating andoften life threatening, there is no effective treatment currentlyavailable.

The human body responds to trauma and injury by scarring. Fibrosis, atype of disorder characterized by excessive scarring, occurs when thenormal wound healing response is disturbed. During fibrosis, the woundhealing response continues causing an excessive production anddeposition of collagen.

Although fibrotic disorders can be acute or chronic, the disorders sharea common characteristic of excessive collagen accumulation and anassociated loss of function when normal tissue is replaced with scartissue.

Fibrosis results from diverse causes, and may be established in variousorgans. Cirrhosis, pulmonary fibrosis, sarcoidosis, keloids,hypertension and kidney fibrosis, are all chronic diseases that induce aprogressive fibrosis which causing a continuous loss of tissue function.

Acute fibrosis (usually with a sudden and severe onset and of shortduration) occurs as a common response to various forms of traumaincluding accidental injuries (particularly injuries to the spine andcentral nervous system), infections, surgery (cardiac scarring followingheart attack), burns, environmental pollutants, alcohol and other typesof toxins, acute respiratory distress syndrome, radiation andchemotherapy treatments. All tissues damaged by trauma are prone to scarand become fibrotic, particularly if the damage is repeated. Deep organfibrosis is often extremely serious because the progressive loss oforgan function leads to morbidity, hospitalization, dialysis, disabilityand even death. Fibrotic diseases or diseases in which fibrosis isevident include pulmonary fibrosis, interstitial lung disease, humanfibrotic lung disease, liver fibrosis, cardiac fibrosis, maculardegeneration, retinal and vitreal retinopathy, myocardial fibrosis,Grave's ophthalmopathy, drug induced ergotism, cardiovascular disease,atherosclerosis/restenosis, keloids and hypertrophic scars, Hansen'sdisease and inflammatory bowel disease, including collagenous colitis.

Further information on different types of fibrosis may be found forexample in Yu et al (2002), “Therapeutic strategies to halt renalfibrosis”, Curr Opin Pharmacol. 2(2):177-81; Keane and Lyle (2003),“Recent advances in management of type 2 diabetes and nephropathy:lessons from the RENAAL study”, Am J Kidney Dis. 41(3 Suppl 2): S22-5;Bohle et al (1989), “The pathogenesis of chronic renal failure”, PatholRes Pract. 185(4):421-40; Kikkawa et al (1997), “Mechanism of theprogression of diabetic nephropathy to renal failure”, Kidney Int Suppl.62:S39-40; Bataller and Brenner (2001), “Hepatic stellate cells as atarget for the treatment of liver fibrosis”, Semin Liver Dis.21(3):437-51; Gross and Hunninghake (2001) “Idiopathic pulmonaryfibrosis”, N Engl J Med. 345(7):517-25; Frohlich (2001) “Fibrosis andischemia: the real risks in hypertensive heart disease”, Am J Hypertens;14(6 Pt 2):194S-199S.

Diabetic Nephropathy

Diabetic nephropathy, hallmarks of which are glomerulosclerosis andkidney fibrosis, is the single most prevalent cause of end-stage renaldisease in the modern world, and diabetic patients constitute thelargest population on dialysis. Such therapy is costly and far fromoptimal. Transplantation offers a better outcome but suffers from asevere shortage of donors. More targeted therapies against diabeticnephropathy (as well as against other types of kidney pathologies) arenot developed, since molecular mechanisms underlying these pathologiesare largely unknown. Identification of an essential functional targetgene that is modulated in the disease and affects the severity of theoutcome of diabetes nephropathy has a high diagnostic as well astherapeutic value.

It is known in the art that many pathological processes in the kidneyeventually culminate in similar or identical morphological changes,namely glomerulosclerosis and fibrosis. Human kidney disease may evolvefrom various origins including glomerular nephritis, nephritisassociated with systemic lupus, cancer, physical obstructions, toxins,metabolic disease and immunological diseases, all of which culminate inkidney fibrosis. The meaning of this phenomenon is that different typesof insults converge on the same single genetic program resulting in twohallmarks of fibrosis: the proliferation of fibroblasts andoverproduction by them of various protein components of connectivetissue. In addition, thickening of the basal membrane in the glomeruliaccompanies interstitial fibrosis and culminates in glomerulosclerosis.Genes encoding proteins that are involved in kidney fibrosis andglomerulosclerosis may be roughly divided into two groups:

1. Genes, the expression of which leads to the triggering ofproliferation of fibroblasts and overproduction by them of variousprotein components of connective tissue. These may be specific todifferent pathological conditions; and2. Genes, the expression of which leads to the execution of the“fibrotic or sclerotic programs”. These may be common to all renalpathologies leading to fibrosis and glomerulosclerosis.

The identification of genes that belong to the second group shouldcontribute to the understanding of molecular mechanisms that accompanyfibroblast and mesangial cell proliferation and hypersecretion, and mayconstitute genetic targets for drug development, aimed at preventingrenal failure. Application of such drugs is expected to suppress,retard, prevent, inhibit or attenuate progression of fibrosis andglomerulosclerosis.

Combination Therapy

The present invention provides for combination therapy for proliferativedisease as disclosed herein and in particular cancer. In saidcombination therapy, one or more genes are targeted to amelioratesymptoms of the disease being treated. These genes are inhibited theantibody-nucleotide complex of the present invention.

Kits

Kits comprising at least one anti-ENDO180 monoclonal antibody of theinvention are further provided. A “kit” refers to any manufacture (e.g.,a package or a container) comprising at least one reagent, i.e., anantibody, for specific binding to ENDO180. The kit may be used forperforming the methods of the present invention, including therapeutictreatment and diagnostics Additionally, the kit may contain a packageinsert describing the kit, its content and methods for use.

In one embodiment, a kit of the invention comprises at least compositioncomprising an anti-ENDO180 antibody or antigen binding fragment thereofselected from

-   -   a) the monoclonal antibody produced by the hybridoma cell line        E3-8D8 (BCCM Accession Number LMBP 7203CB);    -   b) an antibody or fragment thereof that binds to the same        epitope as the antibody in (a);    -   c) a fragment of an antibody comprising a polypeptide        substantially similar to SEQ ID NO: 6; and    -   d) a recombinant polypeptide comprising CDRs having an amino        acid sequence substantially similar to amino acid sequences set        forth in SEQ ID NO:7 and 8.

The invention has been described in an illustrative manner, and it is tobe understood that the terminology which has been used is intended to bein the nature of words of description rather than of limitation.

Citation of any document herein is not intended as an admission thatsuch document is pertinent prior art, or considered material to thepatentability of any claim of the present application. Any statement asto content or a date of any document is based on the informationavailable to applicant at the time of filing and does not constitute anadmission as to the correctness of such a statement.

EXAMPLES General Methods in Molecular Biology

Standard molecular biology techniques known in the art and notspecifically described were generally followed as in Sambrook et al.,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, New York (1989), and in Ausubel et al., Current Protocols inMolecular Biology, John Wiley and Sons, Baltimore, Md. (1989) and inPerbal, A Practical Guide to Molecular Cloning, John Wiley & Sons, NewYork (1988), and in Watson et al., Recombinant DNA, Scientific AmericanBooks, New York and in Birren et al (eds) Genome Analysis: A LaboratoryManual Series, Vols. 1-4 Cold Spring Harbor Laboratory Press, New York(1998) and methodology as set forth in U.S. Pat. Nos. 4,666,828;4,683,202; 4,801,531; 5,192,659 and 5,272,057 and incorporated herein byreference. Polymerase chain reaction (PCR) was carried out generally asin PCR Protocols: A Guide To Methods And Applications, Academic Press,San Diego, Calif. (1990). In situ (In cell) PCR in combination with FlowCytometry can be used for detection of cells containing specific DNA andmRNA sequences (Testoni et al., 1996, Blood 87:3822.)

General methods in immunology: Standard methods in immunology known inthe art and not specifically described are generally followed as inStites et al (eds), Basic and Clinical Immunology (8th Edition),Appleton & Lange, Norwalk, Conn. (1994) and Mishell and Shiigi (eds),Selected Methods in Cellular Immunology, W.H. Freeman and Co., New York(1980).

Immunoassays: ELISA immunoassays are well known to those skilled in theart. Both polyclonal and monoclonal antibodies can be used in theassays. Where appropriate, other immunoassays such as radioimmunoassays(RIA) can be used as are known to those skilled in the art. Availableimmunoassays are extensively described in the patent and scientificliterature. See, for example, U.S. Pat. Nos. 3,791,932; 3,839,153;3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654;3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219;5,011,771 and 5,281,521 as well as Sambrook et al., Molecular Cloning: ALaboratory Manual, Cold Springs Harbor, N.Y., 1989.

Antibody Production

By the term “antibody” as used in the present invention is meant bothpolyclonal and monoclonal complete antibodies as well as fragmentsthereof, such as Fab, F(ab′)2, scFv and Fv, which are capable of bindingthe epitope determinant. These antibody fragments retain the ability toselectively bind with its antigen or receptor and are exemplified asfollows, inter alia:

A Fab, the fragment which contains a monovalent antigen-binding fragmentof an antibody molecule can be produced by digestion of whole antibodywith the enzyme papain to yield a light chain and a portion of the heavychain;A (Fab′)₂, the fragment of the antibody that can be obtained by treatingwhole antibody with the enzyme pepsin without subsequent reduction;F(ab′2) is a dimer of two Fab fragments held together by two disulfidebonds;A Fv, defined as a genetically engineered fragment containing thevariable region of the light chain and the variable region of the heavychain expressed as two chains; andA scFv fragment (i.e. a single chain antibody (“SCA”), defined as agenetically engineered molecule containing the variable region of thelight chain and the variable region of the heavy chain linked by asuitable polypeptide linker as a genetically fused single chainmolecule.

Such fragments having antibody functional activity can be prepared bymethods known to those skilled in the art (Bird et al. (1988) Science242:423-426)

Conveniently, antibodies may be prepared against an immunogen or portionthereof, for example, a synthetic peptide based on the sequence, orprepared recombinantly by cloning techniques or the natural gene productand/or portions thereof may be isolated and used as the immunogen.Immunogens can be used to produce antibodies by standard antibodyproduction technology well known to those skilled in the art, asdescribed generally in Harlow and Lane (1988), Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., andBorrebaeck (1992), Antibody Engineering—A Practical Guide, W.H. Freemanand Co., NY.

For producing polyclonal antibodies a host, such as a rabbit or goat, isimmunized with the immunogen or immunogen fragment, generally with anadjuvant and, if necessary, coupled to a carrier; antibodies to theimmunogen are collected from the sera. Further, the polyclonal antibodycan be absorbed such that it is monospecific; that is, the sera can beabsorbed against related immunogens so that no cross-reactive antibodiesremain in the sera, rendering it monospecific.

For producing monoclonal antibodies the technique involveshyperimmunization of an appropriate donor with the immunogen, generallya mouse, and isolation of splenic antibody-producing cells. These cellsare fused to an immortal cell, such as a myeloma cell, to provide afused cell hybrid that is immortal and secretes the required antibody.The cells are then cultured, in bulk, and the monoclonal antibodiesharvested from the culture media for use.

For producing recombinant antibody see generally Huston et al. (1991)“Protein engineering of single-chain Fv analogs and fusion proteins” inMethods in Enzymology (J J Langone, ed., Academic Press, New York, N.Y.)203:46-88; Johnson and Bird (1991) “Construction of single-chain Fvbderivatives of monoclonal antibodies and their production in Escherichiacoli in Methods in Enzymology (J J Langone, ed.; Academic Press, NewYork, N.Y.) 203:88-99; Mernaugh and Mernaugh (1995) “An overview ofphage-displayed recombinant antibodies” in Molecular Methods In PlantPathology (R P Singh and US Singh, eds.; CRC Press Inc., Boca Raton,Fla.:359-365). Additionally, messenger RNAs from antibody-producingB-lymphocytes of animals, or hybridoma can be reverse-transcribed toobtain complementary DNAs (cDNAs). Antibody cDNA, which can be full orpartial length, is amplified and cloned into a phage or a plasmid. ThecDNA can be a partial length of heavy and light chain cDNA, separated orconnected by a linker. The antibody, or antibody fragment, is expressedusing a suitable expression system to obtain recombinant antibody.Antibody cDNA can also be obtained by screening pertinent expressionlibraries.

The antibody can be bound to a solid support substrate or conjugatedwith a detectable moiety or be both bound and conjugated as is wellknown in the art. (For a general discussion of conjugation offluorescent or enzymatic moieties see Johnstone & Thorpe (1982),Immunochemistry in Practice, Blackwell Scientific Publications, Oxford).The binding of antibodies to a solid support substrate is also wellknown in the art (for a general discussion, see Harlow & Lane (1988)Antibodies: A Laboratory Manual, Cold Spring Harbor LaboratoryPublications, New York; and Borrebaeck (1992), Antibody Engineering—APractical Guide, W.H. Freeman and Co.). The detectable moieties or labelcontemplated with the present invention include, but are not limited to,fluorescent, metallic, enzymatic and radioactive markers such as biotin,gold, ferritin, alkaline phosphatase, β-galactosidase, peroxidase,urease, fluorescein, rhodamine, tritium, 14C and iodine.

Recombinant Protein Purification

For standard purification, see Marshak et al. (1996), “Strategies forProtein Purification and Characterization. A laboratory course manual.”CSHL Press.

The polynucleotide sequence of human ENDO180 mRNA is set forth inaccession number NM_(—)006039: 5641 bases, of that the open readingframe (ORF) is 4439 bases (from 117-4441); the polypeptide sequence of1479 amino acids (aa) is set forth in accession number NP_(—)006030 withgene identifier number: GI:110624774. The mouse mRNA sequence is 5818bases, accession number MMU56734 with ORF of 1479 aa.

ENDO180 comprises several protein domains, as follows: 1-31 aa SP(signal peptide); 41-161 aa cysteine rich N-terminal domain; 180-228 aaFNII (fibronectin type II) domain; 8 CDR (carbohydrate recognitiondomain) domains 1CRD-8CRD (235-360 aa 1CRD, 382-505 aa 2CRD, 521-645 aa3CRD, 669-809 aa 4CRD, 825-951 aa 5CRD, 972-1108 aa 6CRD, 1161-1244 aa7CRD, 1261-1394 aa 8CRD); 1413-1435 aa 1 TM (transmembrane domain),1437-1479 α-cytoplasmic domain.

Reference to Sequence Listing

The sequences described in the specification (SEQ ID NOS:1-9) are beingsubmitted with this application via the USPTO electronic filing system(EFS) in a text file titled, “202-PCT1.5T25.txt” created on Mar. 23,2010 file size 36 KB, and are hereby incorporated by reference herein intheir entirety.

Example 1 Identification of ENDO180 Overexpression by MicroarrayHybridization Study

In accordance with the present invention, the microarray hybridizationapproach was utilized in order to discover genes that are differentiallyregulated in diabetic nephropathy and kidney fibrosis.

Microarray-based analysis of gene expression was based on the analysisof human fibroblasts subject to selected stimuli resulting in changes inextracellular collagen accumulation and proliferation—the hallmarks offibrosis. According to the present invention, a specific “Fibrosis” DNAchip was first prepared followed by a microarray hybridizationexperiments with 19 different types of probes. Analysis of the resultswas carried out by proprietary algorithms, and analysis of the selectedset of genes was performed by the inventors using bioinformatics and thescientific literature.

Example 2 Production of Human Anti-ENDO180 Antibodies

The aim was to generate anti-ENDO180 antibodies that bind to theextracellular portion of ENDO180 and internalize an anti-ENDO180antibody-cargo complex/conjugate.

Antigen production: The structural considerations in selecting anantigen for antibody production included the information that aminoacids 1-522 (SEQ ID NO:9) spatially create the ligand binding structure.

Recombinant ENDO180 antigen was produced by cloning nucleotides 1-1566of human ENDO180 polynucleotide into an expression vector comprising theFLAG epitope. The polynucleotide sequence of the recombinant clone isset forth in SEQ ID NO:3, the amino acid sequence is set forth in SEQ IDNO:4. The vectors were transfected into 293T cells and a clonesexpressing the 59 KD partial extracellular domain of human ENDO180 wereidentified. The ECDhENDO180-FLAG protein was isolated as follows: about2.2 liters of conditioned medium were filtered through a 0.22 um filter.Medium was loaded on a pre-equilibrated (with TBS) M2 agarose (5 ml,Sigma) at a flow rate of 1 ml/min. Resin was washed with 10 columnvolumes using TBS and then 10 volumes with 50 mM Tris pH 7.5, 1M NaCland finally with 10 volumes of TBS. Elution was done with 10 ml of 0.5mg/ml FLAG peptide in TBS pH 7.5 (final pH). Resin was incubated for 20min with elution buffer before starting the flow out of column. Samplewas concentrated and depleted of FLAG peptide using VivaSpin™ (cut-off10 Kd). Glycerol was added to 10% final and protein was flash frozen inliquid nitrogen.

Identification of minibodies: minibody antibodies were identifiedaccording to the methods disclosed in Di Niro et al, 2007. Constructionof miniantibodies for the in vivo study of human autoimmune diseases inanimal models. BMC Biotechnology 7:46.

Certain preferred antibodies according to the present invention arerecombinant polypeptides comprising heavy chain and light chain CDR3domains having amino acid sequences set forth in SEQ ID NO:7 and in SEQID NO:8.

Example 3 Anti-ENDO180 Monoclonal Antibodies Methods and Summary:

-   -   1. Labeling of mAbs and MB with CypHer5e was performed according        to manufacturer's directions. (GE Healthcare).    -   2. Labeled vs. unlabeled mAbs and MB were tested for binding to        purified ENDO180 extra-cellular domain by standard ELISA.        -   Clones 6D6, 8D8, 8E7 and 9G10 displayed saturated binding to            endo180DCTLD3-8-FLAG even after labeling with CypHer5E. In            contrast, binding of clone 8D2 and MB (minibody) was            significantly impaired upon labeling.    -   3. Internalization assays were performed according to methods to        those skilled in the art.        -   After 1 hr at 37° C., ENDO180 expressing cells that were            incubated with labeled mAbs 6D6, 8D8, 8E7 and 9G10,            displayed some increase in fluorescence. Most notably, the            same cells that were incubated with labeled MB, showed            strong fluorescence. This increase in fluorescence was not            seen in control cells or in ENDO180 cells at 40 C. In            addition, control mAb (10F12) had no effect    -   4. Kinetics experiments        -   Based on the results of previous experiments, mAb 6D6, 8D8            and MB were tested. In the 10 min-1 hr kinetics experiment,            mAb 8D8 and MB showed the best internalization. All negative            controls, control cells, (4° C.; shown as 4OC in some            figures) and control mAb-were negative.

Details of Experiments and Results

Anti-ENDO180 monoclonal antibodies (mAb) were generated against the mostN-terminal domain (1-522 aa) of human ENDO180 (SEQ ID NO:9) and werescreened for internalization in an ENDO180-specific manner per se andconjugated to the fluorophore, CypHer5E.

mAbs production: About 8 liters of each hybridoma were grown in DMEMmedium supplemented with 5% FBS IgG FREE, 1% Penstrep, 1% L-Glutamine,50 μg/ml Gentamycin, 2.5 μg/ml Amphotricin B (Fungizone). About 60 mlwas obtained from cell line flasks, with an antibody concentration ofabout 200 μg/ml. The duration of the growth was 1 month. Thepurification was done using Protein A Sepharose column followed by twocycles of sizing column.

The mAb 8D8 (E3-8D8), 6D6, 8E7, 9G10, 8D2 were selected for conjugationto labeling moieties.

The mAbs were covalently linked to CypHer5E (Cat #pA15405, Amersham) ared excited fluorescent pH sensitive cyanine dye according tomanufacturer's instructions in a molar ratio of 20:1 CypHer5E:Antibody.The fluorophore is excited in acidic pH, as found within the endosome.Therefore, those antibodies that get internalized will be seen asfluorescent signals in cellular vesicles. The mAbs were covalentlylinked to biotin using EZ-Sulfo-NHS-biotin (Pierce, Cat #21217, Lot#CE49927). Biotinylation was performed according to manufacturer'sinstructions using a molar ratio of 20:1 Biotin:Antibody at roomtemperature for 30 min. Following covalently binding of mAbs to CypHer5Eor biotin, the antibody solution was dialyzed overnight at 4° C. againstsolution of PBS following additional two hours at room temperatureagainst a fresh PBS solution. Labeling of mAbs and scFv (SEQ ID NO. 6,also referred to as “minibody”) with CypHer5E was performed according tomanufacture's instructions (GE Healthcare).

Internalization of the conjugated receptor was performed in the absenceof collagen (ligand independent internalization). The following cloneswere shown to secrete antibodies that bind specifically to ENDO180:Clones 6D6, 8D8, 8E7 and 9G10 displayed saturated binding toendo180DCTLD3-8-FLAG per se and when labeled with CypHer5E. Clone 8D2showed limited uptake into cells following labeling while clone 10F12exhibited significant and saturated binding to endo180DCTLD3-8-FLAGwhich was diminished after labeling.

Cells expressing ENDO180 (NRK52E-ENDO180) and control cells (NRK52E)were incubated at 37° C. with the indicated anti-ENDO180 mAbs or controlmAbs, covalently linked to CypHer5E. The mAbs were also incubated at 4°C. with ENDO180 cells. The cells were plated in a 96-well plate andfluorescence was measured by Analyst AD &HT, Biosystems (excitation 530nm, emission 590 nm, dichroic 560 nm). A steady increase in fluorescenceat 37° C. in ENDO180 cells was seen with one mAb (E3-8D8). In contrast,fluorescence was not seen in control cells, at 4° C. or with controlmAbs. Antibody binding and internalization was tested in ENDO180expressing NRK52 cells at permissive (37° C.) and non-permissive (4° C.)temperature and tested after one hour. FIG. 2A shows level offluorescence in ENDO180 expressing calls at permissive (37° C.) andnon-permissive (4° C.). Clones 6D6 and 8D8 and the scFv (SEQ ID NO:6)show highest level of internalization. FIG. 2B shows that 8D8 exhibitsno non-specific binding.

The ENDO180 receptor was shown to be an internalization and recyclingreceptor (Howard and Isacke, 2002, JBC 277, 35:32320-31) yet not allantibodies produced are internalized at the same rate or in the sameamount. mAb 8D8 and the G7V scFv showed unexpected internalization. NomAb 10F2 was internalized, even after 8 hours.

Kinetics of internalization was studied: NRK52 cells stably expressinghuman ENDO180-FLAG or empty vector, were seeded in TC-grade black96-well plates at a density of 6000 cells/well. At 24 or 48 hrs later,mAbs (5 ug/ml in growth medium) were added to the wells, 100 ul/well ateither room temperature or on ice (for control plates). The plates wereimmediately incubated at 37° C. for various times. Control plates werekept on ice for the required times. At each time point, the plates werewashed 3× in 200 ul ice-cold PBS. After last wash, 100 ul ice-cold PBSwas added and plates read using Analyst at Ex 610 nm/Em 670 nm.

FIGS. 2C-2E show a 10-minute to 1-hour time course of internalization ofanti-ENDO180-CypHer5E conjugates by ENDO180 expressing cells. FIG. 2Cshows internalization of CypHer5E labeled 8D8. FIG. 2D showsinternalization of CypHer5E labeled scFv G7V (SEQ ID NO:6). FIG. 2Eshows internalization of CypHer5E labeled 10F2.

FIGS. 2F-2H shows a 1-hour to 8-hour time course of internalization ofanti-ENDO180-CypHer5E conjugates by ENDO180 expressing cells. FIG. 2Fshows internalization of CypHer5E labeled 8D8. FIG. 2G showsinternalization of CypHer5E labeled scFv G7V (SEQ ID NO:6). F1G. 2Eshows internalization of CypHer5E labeled 10F2.

The hybridoma cell line E3-8D8 that secretes monoclonal antibody E3-8D8.also referred to as 8D8, was deposited as per the Budapest Treaty in theBelgian Co-ordinated Collections of Micro-organisms (BCCM); Departmentof Biomedical Molecular Biology; Ghent University with Accession NumberLMBP 7203CB. The deposit was made on 9 Mar. 2010 and tested and shown tobe viable on 18 Mar. 2010.

A composition for the systemic uptake of a drug across a mucosalmembrane comprising a polyethylene glycol-chitosan conjugate, whereinthe polyethylene glycol-chitosan conjugate comprises a chitosan orchitosan derivative moiety and a polyethylene glycol or a polyethyleneglycol derivative moiety, and the composition is formulated for deliveryto a mucosal membrane.

The general method of preparing a substrate-agent conjugate according tothe invention involves covalently binding at least one therapeutic ordiagnostic agent to a substrate. Certain cytotoxic drugs that are usefulfor anticancer therapy are relatively insoluble in serum. Some are alsoquite toxic in unconjugated form and their toxicity is considerablyreduced by conversion to conjugates. Conversion of a relatively poorlysoluble drug to a more soluble conjugate, e.g., a glucuronide, willimprove its solubility in the aqueous phase of serum and its ability topass through venous, arterial or capillary cell walls and reach theinterstitial fluid bathing the tumor. In fact, conversion of certaintoxic substances such as aromatic or alicyclic alcohols, thiols, phenolsand amines to glucuronides in the liver is the body's method ofdetoxifying them and making them more easily excreted in the urine.

Example 4 In Vitro and In Vivo Internalization of Antibody Conjugates

FIG. 3 shows internalization of Biotin by anti-ENDO180 mAbs to miceUnilateral Ureter Obstructed kidney.

The following experiment was designed in order to assay ENDO180 antibodyaccumulation in ENDO180 expressing tissue. Unilateral ureter obstructed(UUO was performed in mice. The level of ENDO180 in kidneys at day 7 ofUUO surgery, was higher than in the contra-lateral kidney (Data notshown).

Mice were injected with 3 mg/kg of E3-8D8-Biotin conjugate orNMIgG-Biotin conjugate at day 7 post UUO surgery, 24 hours later thekidney. The level of E3-8D8-Biotin conjugate and NMIgG-Biotin (normalmouse IgG control) conjugate uptake in the Unilateral ureter obstructed(UUO) kidney and Contra-lateral kidney was examined by Western blot(WB). The same amount of kidney total protein extract was examined by WBusing Goat-anti-Biotin HRP (Cell signaling #7075).

FIG. 4 shows internalization of anti-ENDO180-CypHer5E conjugate inMyelo-Monocytoid human leukemia MonoMac cell line expressing ENDO180.MonoMac cells were incubated with E3 8D8-CypHer5E or E3 8D2-CypHer5E.Cells were washed twice with cold PBS and internalization was measuredby FACS using FL-1 or FL-4 filter. E3 8D8 bound ENDO180 with higheraffinity than E3-8D2 (Data not shown). 8D2 is a mAb that binds ENDO180with lower affinity than 8D8.

Example 5 Linking Antibody to Therapeutic Agent

-   -   a. MB: Full human protamine (˜50 a.a) is cloned directly        downstream to the constant region of the heavy chain. A similar        strategy was taken using anti HIV-1gp 120 recombinant Fab        fragment with a bicistronic vector expressing VH-CH1-protamine        from one promoter and VKCK light chain from another (Chen et        al., Gene Therapy (1995) 2, 116-123). This construct was shown        to have in vivo anti-tumor activity (Song et al., Nature        Biotech. 2005. 23(6), pg. 709-717). In another study, protamine        was fused downstream to single chain antibody to ErbB (Li et        al., Cancer Gene Therapy 8(8), 2001, pg. 555-565).    -   b. mAb: The CDR domains of the monoclonal antibody 8D8 are        sequenced and cloned so that protamine can be engineered in        fusion with it as with the MB.    -   c. Standard methods are used to link the antibody or        antigen-binding fragment thereof (MB, isolated mAb, scFv, F′ ab        etc.) to the therapeutic agent using one or more of a peptide,        nucleic acid, chemical or lipid linker

Example 6 In Situ Hybridization in Cancer Tissue Samples

Samples of human tissue from cancer patients were tested form expressionof ENDO180 using in situ hybridization techniques. The tissue sampleswere analyzed by a skilled pathologist. The results showed the followingexpression patterns:

1. Renal Cell Carcinoma (RCC)

-   -   High level of ENDO180 mRNA expression appeared in cells in all        five sarcomatoid areas studied, in two different renal cell        carcinoma types—clear cell (four cases) and papillary (one case)        carcinomas. Sarcomatoid carcinomas develop in all main types of        renal cell carcinomas (clear cell, papillary, chromophobe and        collecting duct carcinomas). They appear in approximately 1-1.5%        of all adult renal tumors and are associated with an aggressive        clinical course and poor prognosis.    -   ENDO180 mRNA expression appears also in intratumoral stromal        cells, in non tumoral stromal cells and in glomerular cells,        with some sample to sample variation of the amount of cells, and        signal intensity.

2. Ovarian Cancer:

-   -   In most borderline serous papillary tumors that are represented        in this study (7 out of 8), ENDO180 expression appeared in        subsets of tumor cells, in various intensities.    -   ENDO180 mRNA expression in ovarian cancer cells appears in 8 out        of 21 cases with some sample-to-sample variation of both amount        and signal intensity of expressing cells.    -   High intensity signals of ENDO180 mRNA expression appeared in        subsets of peritumor stromal cells and in subsets of ovarian        stromal cells (94% and 100%). ENDO180 mRNA expression was also        detected in single cells in normal epithelium.

3. Transitional Cell Carcinoma (TCC):

-   -   In most primary transitional cell (urothelial) carcinoma of        bladder (18 out of 27 cases), and 4 cases of metastatic (in        lymph nodes) tumors, that are represented in this study, ENDO180        expression was weak. High intensity signals of ENDO180 mRNA        expression appeared in subsets of peritumor stromal cells.

4. Breast Cancer:

-   -   Expression of ENDO180 mRNA was seen in the peritumoral cells in        most of the cases of invasive carcinoma (84%). No consistent        expression pattern in epithelial cells.

Example 7 Animal Models for Testing Compounds in Treatment of Fibrosis

The following animal models are presented as non-limiting examples foruse in testing exemplary molecules and conjugates of the presentinvention for efficacy in treating a subject suffering from fibrosis andfibrotic diseases. Other animal models are also considered.

A useful way to assess the development of renal diseases involvingfibrosis and glomerulosclerosis is to characterize gene expression inestablished animal models of kidney diseases. Examples of such modelsinclude without limitation: (i) fa/fa rats—animals genetically deficientin leptin receptor that develop insulin resistant diabetes (type IIdiabetes) with progressive diabetic nephropathy, and (ii) GK rats—whichare genetically manipulated, NIDDM phenotype rats. Another animal modelin which mainly kidney fibrosis is evident, but without a background ofdiabetes, is unilateral ureteral obstruction (UUO) in which interstitialfibrosis is rapid and occurs within days following the obstruction. 5/6nephrectomy is another useful animal model for chronic renalinsufficiency (CRI) in which fibrosis is evident.

Additional aspects of research may be based on an in vitro model systeminvolving culture of human fibroblasts in vitro under conditionsmimicking various parameters of the cell microenvironment existing inCRI and fibrosis. These include treatment with high concentrations ofglucose (modeling hyperglycemia), low concentrations of glucose, hypoxia(both modeling ischemic conditions that develop in the kidney followingfibrosis and glomerulosclerosis), and TGF-b—one of the recognizedpathogenic factors in fibrosis.

Such in vitro model systems may complement the animal models in severalimportant aspects: First, the system is fibroblast-specific;accordingly, none of the interferences often found in complex tissuesthat contain many cell types are present. Second, the cells are of humanorigin, unlike the animal models. Furthermore, the insults are specificand of various concentrations and duration, thus enabling theinvestigation of both acute and chronic responses.

Example 8 Animal Models for Testing the Compounds in Cancer Therapy

The following animal models are presented as non-limiting examples foruse in testing exemplary molecules and conjugates of the presentinvention for efficacy in treating a subject suffering from cancer andother proliferative and metastatic diseases. Other animal models arealso considered.

Transplantation in Immunodeficient Mice

The NOD/SCID mouse is defective in both lymphoid and myeloid functionand readily accepts the long-term survival of human hematopoietic cells.Transplantation of human bone marrow into NOD/SCID mice to human/mousechimeras, is well documented.

The NOD/SCID mice were used by Bertolini et al (2000. Blood 96-282) as amodel high-grade non-Hodgkin lymphoma. The Namalwa cell line was used,which is derived from an Epstein-Barr virus-positive Burkitt non-Hodgkinlymphoma. The cells (10×10⁶) were injected intraperitoneally into 6-8weeks old mice. Intraperitoneal tumors were formed in the injection sitewhich could be measured by calipers. The formula: “width 2×length×0.52”was applied to approximate the volume of a spheroid. (see Bohem et al(1997) for further reference).

The model used in the following studies is based on transgenic SCID miceexpressing human GM-CSF. The expression of this cytokine enabled thesuccessful grafting of relevant cells lines in the SCID mice. Miyakawaet al (1996) details the production of the hGM-CSF SCID transgenic mice.Fukuchi et al (1998) shows that a retinoic-acid resistant leukemia canbe established in these transgenic mice. The model consisted of UF-1cells, an RA-resistant APL cell line established in that laboratory,which are transplanted into these transgenic SCID mice and cause theappearance of subcutaneous tumors. Kinjo et al (2000) uses this model totest a specific treatment, arsenic trioxide, to be used in cases of RAresistant acute promyelocytic leukemia (APL).

Lewis et al (1998) used the more profoundly immunodeficient mouse strainNOD/SCID in which both T-cell and B-cells are functionally defective,and there is marked impairment of macrophage, natural killer cell, andhemolytic complement activity. These mice can be engrafted with cellstaken from cancer patients leading to a relatively high success rate andthus form a good model for the disease.

1. An anti-ENDO180 antibody or antigen-binding fragment thereof selectedfrom the group consisting of a. an isolated monoclonal antibody producedby the hybridoma cell line E3-8D8, deposited with the BCCM underAccession Number LMBP 7203CB; b. an antibody or a fragment thereof thatbinds to the same epitope as the antibody of (a); c. a humanized versionof the antibody of (a) or a humanized antibody or fragment of (b); andd. a chimeric version of the antibody of (a) or a chimeric antibody orantigen-binding fragment wherein upon contact with a cell expressingENDO180, the antibody, or the antigen binding fragment, is internalizedinto the cell.
 2. A composition comprising the anti-ENDO180 antibody orthe antigen binding fragment of claim 1, and a pharmaceuticallyacceptable carrier.
 3. The composition of claim 2, wherein theanti-ENDO180 antibody or the antigen binding fragment further comprisesa moiety selected from the group consisting of a detectable label, acytotoxic agent and a therapeutic agent.
 4. The composition of claim 2wherein the carrier comprises a lipid particle, a polysaccharideparticle or a combination thereof.
 5. The composition of claim 4,wherein the carrier comprises a lipidated polysaccharide particle. 6.The composition of claim 5 wherein the lipidated polysaccharide particlecomprises a lipidated glycosaminoglycan particle.
 7. The composition ofclaim 4, wherein the anti-ENDO180 antibody or the antigen-bindingfragment is immobilized on the particle.
 8. The composition of claim 3,wherein the moiety is a therapeutic agent.
 9. The composition of claim8, wherein therapeutic agent is an inhibitory oligonucleotide.
 10. Thecomposition of claim 9, wherein the inhibitory oligonucleotide isselected from the group consisting of antisense compounds, chemicallymodified siRNA compounds, unmodified siRNA compounds, chemicallymodified shRNA compounds, unmodified shRNA compounds, chemicallymodified miRNA compounds, and unmodified miRNA compounds.
 11. Thecomposition of claim 10, wherein the inhibitory oligonucleotide is achemically modified siRNA compound.
 12. The composition of claim 11,wherein the chemically modified siRNA compound inhibits expression of atarget gene associated with cancer, fibrosis or a macrophage associateddisease. 13-14. (canceled)
 15. The composition of claim 3, wherein themoiety is encapsulated within the lipid particle. 16-20. (canceled) 21.A hybridoma cell line designated E3-8D8, deposited with the BCCM underAccession Number LMBP 7203CB. 22-25. (canceled)
 26. The composition ofclaim 3, wherein the antibody or the antigen-binding fragment isconjugated to said moiety directly or via a linker which links theantibody or the antigen-binding fragment to the moiety.
 27. Thecomposition of claim 26 comprising a linker, wherein the linker attachesthe 3′ terminus of the antibody or the antigen-binding fragment to themoiety.
 28. (canceled)
 29. The composition of claim 27, wherein thelinker is selected from the group consisting of polypeptide linkers,peptide linkers, lipid linkers and nucleic acid linkers. 30-42.(canceled)
 43. A method of treating a subject afflicted with cancer,fibrosis or a macrophage associated disease comprising administering tothe subject a composition of claim 8 in an amount effective to treat thesubject. 44-45. (canceled)
 46. The antibody or the antigen-bindingfragment of claim 1, wherein the antibody or the antigen-bindingfragment is selected from the group consisting of full IgGs, Fabfragments, Fab′ fragments, F(ab′)2 fragments, variable portions of heavychains or light chains any of the preceding, Fab miniantibodies, andscFvs. 47-80. (canceled)